Virus-like particles comprising zika antigen

ABSTRACT

The invention is related to chimeric Virus-Like Particles (VLPs) containing and displaying epitopes and antigen from Zika Virus (ZIKV); and to methods for creation and production of such chimeric VLPs to their applications, including but not limited to vaccines, diagnostics, clinical studies, assay development and antibody discovery.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a § 371 national stage application of InternationalApplication No. PCT/US2018/039079, filed Jun. 22, 2018, which claims thepriority benefit under 35 U.S.C. § 119(e) of U.S. Provisional PatentApplication No. 62/524,440, filed Jun. 23, 2017, the disclosure of whichis incorporated herein by reference in its entirety. The entiredisclosure of each of the aforesaid applications is incorporated byreference in the present application.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of the disclosure, aSequence Listing in computer-readable form which is incorporated byreference in its entirety and identified as follows: Filename:52049A_Seqlisting.txt; Size-123,456 bytes, created: Jun. 22, 2018.

FIELD OF THE INVENTION

This invention is related to improved tools for detection of Zika Virus(ZIKV), ZIKV vaccines, and ZIKV diagnostics.

BACKGROUND

Zika virus (ZIKV) is an arbovirus belonging to the Flavivirus genus.ZIKV was first isolated from an infected sentinel monkey in the Zikaforest in Uganda (1947), and later in mosquitos [1, 2] and humans in1954 [3]. No outbreaks were described until 2007 when a ZIKV epidemic onthe Island of Yap in the Federated States of Micronesia showed that thevirus had the propensity to cause serious disease [4, 5]. Subsequently,ZIKV spread to French Polynesia and Pacific Islands (2013-2014), andrecently to the Americas causing very large outbreaks in more thantwenty countries including Brazil, Mexico and the Caribbean Islands(2015-present). There is evidence that ZIKV transmission can also occursexually [6, 7], by blood transfusion and possibly via placenta toinfect the fetus [8].

ZIKV is transmitted by mosquitoes of the widely distributed speciesAedes aegypti and Aedes albopictus [4, 5]. According to the Centers forDisease Control (CDC), Aedes mosquito species are distributed in manyterritories of the United States harboring either subtropical ortemperate climates. Indeed, ZIKV has caused multiple local infections inthe US and US territories, including Puerto Rico, Florida and Texas [9,10]. ZIKV may continue to spread globally and be introduced in Europeand Australia, and is likely to reemerge in Africa and Asia. ZIKVinfection is asymptomatic in a majority (approximately 80%) of peopleexposed to the virus. Symptoms of infection are similar to otherarbovirus diseases, such as Dengue virus (DENV) and Chikungunya Virus(CHIKV), and include fever, maculopapular rash, conjunctivitis, andarthralgia, confounding accurate diagnosis. Importantly, there is astrong association of ZIKV with the autoimmune disease, Guillain-BarréSyndrome (GBS), and congenital malformations resulting in Microcephaly[4, 11].

To date, no prophylactic or therapeutic treatment is commerciallyavailable and licensed for ZIKV, despite intensive efforts in thisdirection.

SUMMARY

The present invention includes a novel ZIKV virus like particle (VLP)and materials and methods for making and using such particles, includingformulations and uses as a vaccine, a prophylactic, therapeutic, anddiagnostic.

The development of a safe and effective vaccine to protect against ZIKVinfection is a high priority objective to reduce the incidence andspread of the severe forms of the disease. An urgent need for thevaccine is also underlined by the impact of the infection on pregnantwomen and the still unknown implications to men who may become infected.

An ideal vaccine candidate, in addition to having a high safety profile,should also be cost effective and economically viable with ease of largescale manufacture. Live virus vaccines and inactivated virus vaccinesare expensive to manufacture due the requirement of highly stringentprocesses and containment facilities (BSL2 or BSL3), using sophisticatedbiological production systems (e.g., mammalian cells, eggs). Animportant caveat with an attenuated vaccine is the safety profile,particularly due to the potential for reversions that mayreduce/eliminate attenuation. In the context of a virus like ZIKV,especially if required to be administered to pregnant women, this willbe a critical concern.

In the case of inactivated whole virus vaccines, often the inactivationmethodologies render epitopes ineffective. Epitope stability isimportant to the production of completely neutralizing antibodies orprotective antibodies.

With the unresolved issue of the role of non-neutralizing andcross-reacting Dengue antibodies enhancing ZIKV infection, similarphenomena may be anticipated in the case of non-neutralizing orpartially neutralizing cross-reacting ZIKV antibodies. With theprevalence of diverse strains of ZIKV, and pending information oneffective cross-protection between diverse strains, a VLP strategyengineered using highly conserved regions of ZIKV surface glycoproteins,as provided herein, provides significant advantages. This approach willmaintain the ZIKV epitope architecture while increasing the possibilityof cross-protection across multiple ZIKV strains and cost-effectivescale-up. During the last decade, advances in VLP production,purification, and adjuvant optimization led to several licensed vaccinesfor viral diseases [12]such as human papilloma virus (HPV), hepatitis Bvirus (HBV), hepatitis E virus (HEV), and influenza. VLPs are moreefficient for stimulating the immune system with respect to the subunitproteins because they have the ability to mimic the native morphology ofthe target virion and they display a repetitive array of epitopes inhigh concentration. In addition, VLPs are safe due to the absence ofreplicating viral genetic material [12].

In some aspects, the VLPs disclosed herein are able to cross-protectacross different strains of ZIKV. Contrary to other vaccinationstrategies such as live attenuated vaccines, a VLP strategy as disclosedherein possesses a higher safety profile, particularly for high riskpopulations such as immunocompromised individuals and pregnant women. Incontrast to purified protein vaccines, VLPs of the disclosure expressthe immunological entity in higher concentration, in an appropriateconfirmation (folding) that expresses the epitopes effectively, and witha higher stability profile. From a product development perspective, thetechnology disclosed herein will also lend itself to facilitatedscale-up with defined quality control strategies for large scaleproduction.

Accordingly, in some aspects the invention includes isolated peptidessuitable for making Zika vaccines or Zika antibodies or for detectingZika antibodies. For example, the invention includes an isolated peptideor protein comprising or consisting of an amino acid sequence that is atleast 80% identical to a sequence as set out in any one or more of SEQID NOs: 2-11, 22-33, 46-47, or 50-51. Genera of peptides with higherminimum percent identity, including 85%, 86%0, 87%, 88%, 89%, 90%0, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% minimum identity to a referencesequence also are contemplated. In some embodiments, the isolatedpeptide or protein comprises or consists of an amino acid sequence thatis 100% identical to a sequence as set out in any one or more of SEQ IDNOs: 2-11, 22-33, 46-47, or 50-51, or an immunogenic fragment thereof.In some aspects, the disclosure provides an isolated peptide comprisingor consisting of an amino acid sequence that is at least 80% identicalto a sequence as set out in any one or more of SEQ ID NOs: 2-11. In someembodiments, the isolated peptide comprises or consists of an amino acidsequence that is 100% identical to a sequence as set out in any one ormore of SEQ ID NOs: 2-11. In some aspects, the disclosure provides anisolated peptide comprising or consisting of an amino acid sequence thatis at least 80% identical to a sequence as set out in any one or more ofSEQ ID NOs: 22-33. In some embodiments, the isolated peptide comprisesor consists of an amino acid sequence that is 100% identical to asequence as set out in any one or more of SEQ ID NOs: 22-33. In someaspects, the disclosure provides an isolated peptide comprising orconsisting of an amino acid sequence that is at least 80% identical to asequence as set out in any one or more of SEQ ID NOs: 46-47. In someembodiments, the isolated peptide comprises or consists of an amino acidsequence that is 100% identical to a sequence as set out in any one ormore of SEQ ID NOs: 46-47. In some aspects, an isolated peptide isprovided comprising or consisting of an amino acid sequence that is atleast 80% identical to a sequence as set out in any one or more of SEQID NOs: 50-51. In some embodiments, the isolated peptide comprises orconsists of an amino acid sequence that is 100% identical to a sequenceas set out in any one or more of SEQ ID NOs: 50-51.

In related aspects, the invention includes a chimeric peptide comprisinga peptide derived from Zika as described herein, including those of thepreceding paragraph, linked to a heterologous peptide or protein havingan amino acid sequence that is at least 90% identical to a WoodchuckHepatitis core Antigen protein (WHcAg) comprising or consisting of anamino acid sequence as set out in SEQ ID NO: 1, or comprising orconsisting of at least one fragment of SEQ ID NO: 1. In some variations,the Zika-derived peptide portion of the chimeric peptide is insertedwithin the WHcAg-derived portion. Higher minimum percent identities alsoare contemplated, as described in the preceding paragraph. In someembodiments, the chimeric peptide further includes at least one peptidelinker of 1-10 amino acids linking the sequence that is at least 80%identical to any one or more of SEQ ID NOs: 2-11 or 22-33 or 46-47 or50-51 to the sequence that is at least 90% identical to a WHcAg protein.In some embodiments, the chimeric peptide comprises or consists of anamino acid sequence at least 90% or at least 95% identical to any one ofSEQ ID NOs: 12-21, 34-45, 48-49, and 52-53.

For example, the invention includes a chimeric peptide comprising aZika-derived peptide as described herein linked to a heterologouspeptide having an amino acid sequence that is at least 90% identical toa Woodchuck Hepatitis core Antigen protein (WHcAg) comprising orconsisting of an amino acid sequence as set out in SEQ ID NO: 1, whereinthe Zika-derived peptide is inserted into the WHcAg-derived peptide at aposition between amino acids 77 and 82 of SEQ ID NO: 1.

In related aspects, the invention includes a polynucleotide comprising anucleotide sequence encoding any of the polypeptides described herein.For example, the invention includes a polynucleotide that comprises anucleotide sequence that encodes a chimeric peptide described herein.Polynucleotides that are DNA, RNA, and that comprises synthetic ormodified nucleotides are contemplated. In some embodiments, thepolynucleotide comprises a nucleotide sequence at least 90% identical toor at least 95% identical to any one of SEQ ID NOs: 65-74, 87-98,101-102, and 105-106.

The invention further includes, in some aspects, a vector comprising apolynucleotide as disclosed herein. In some variations, the vectorcomprises an expression vector comprising a polynucleotide of thedisclosure operably linked to an expression control sequence. Vectorssuitable for expression in all varieties of host cells are contemplated,including prokaryotic expression vectors and eukaryotic expressionvectors. Exemplary eukaryotic expression vectors include vectors forexpression in mammalian cells, insect cells, plant cells, avian cells,amphibian cells, and fungal cells, including yeast cells.

In further aspects, the invention includes a recombinant host cellcomprising a vector or expression vector as disclosed herein. In someembodiments, the host cell is (a) a eukaryotic cell selected from thegroup consisting of mammalian, fungal (e.g., yeast), insect, plant,amphibian and avian cells; or (b) a prokaryotic cell.

The invention also includes a composition comprising a vector asdescribed herein in a pharmaceutically acceptable carrier, diluent,stabilizer, preservative, or adjuvant.

In some aspects, the invention includes a virus-like particle (VLP)comprising or consisting essentially of one or more chimeric peptides orproteins described herein. In further aspects, the invention includes anarticle comprising a chimeric peptide or protein as described herein ora VLP as described herein attached to a solid support. In someembodiments, the solid support is a microbead, an assay plate, a teststrip, or a filter. In some variations, the article further includes adistinct peptide attached to the article, optimally at a spatiallydistinct location, that can serve as a positive or negative control inassays described herein. In still other variations, the article ispackaged as part of a kit with at least one assay reagent, such as animmunoassay reagent.

The invention also includes a composition comprising a peptide orprotein described herein, or a chimeric peptide or protein describedherein, and a pharmaceutically acceptable diluent, adjuvant, excipient,stabilizer, preservative, or carrier. In some variations, the disclosureprovides an antigenic composition comprising a VLP as disclosed herein,wherein the VLP is present in the composition at a concentration ofabout 0.1-2000 μg/ml of core antigen, in a pharmaceutically acceptablecarrier, diluent, stabilizer, preservative, or adjuvant.

In still additional variations, the invention includes an antigeniccomposition comprising two or more different polypeptides, chimericpolypeptides, or VLP's described herein. For instance, the inventionincludes first and second VLP described herein, wherein the first andsecond VLP comprise different sequences independently selected fromamino acid sequences at least 80% identical to SEQ ID NOs: 2-11, 22-33,46-47, and 50-51. For embodiments of this nature, ordinals such as“first” or “second” are intended simply to differentiate one fromanother, and are not intended to imply an order. Such compositionsoptionally further includes a pharmaceutically acceptable diluent,adjuvant, excipient, stabilizer, preservative, or carrier.

In a related embodiment, the invention is an antigenic compositioncomprising first, second, and third VLP as described herein. Forinstance, the first, second, and third VLP comprise different sequencesindependently selected from amino acid sequences at least 80% identicalto SEQ ID NOs: 2-11, 22-33, 46-47, and 50-51.

In another related embodiment, the invention is an antigenic compositioncomprising first, second, third, fourth, fifth, sixth, and seventh VLPas described herein. For example, the first, second, third, fourth,fifth, sixth, and seventh VLP comprise different sequences independentlyselected from SEQ ID NOs: 2-11, 22-33, 46-47, and 50-51.

The invention also includes a kit comprising a VLP as described herein,or comprises an article of manufacture as described herein, packagedwith at least one reagent useful for performing an immunoassay.Exemplary suitable reagents include an enzyme substrate, a detectionantibody, positive and negative control reagents, substrate/s detectionsolution, and washing, blocking, and diluent buffer. In someembodiments, the kit includes a specific apparatus used for theexecution of the protocol and for the detection.

The invention further includes an antigenic composition comprising apeptide, a chimeric peptide or chimeric protein, or a VLP as describedherein in a pharmaceutically acceptable carrier, diluent, stabilizer,preservative, or adjuvant, wherein the composition is capable ofgenerating an immune response to a Zika virus. An exemplary immuneresponse includes antibody generation or a protective immune response ina mammalian subject. Desirably, the antibody response generated by thecomposition is improved relative to or compared to an immune responseachieved with live Zika virus or Zika Envelope (E) recombinant protein.In some variations, the antibody response is a protective and functionalagainst Zika virus by neutralizing activity, and/or antibody dependentcell-mediated cytotoxicity (ADCC), and/or antibody dependentcell-mediated phagocytosis (ADCP), and/or complement-dependentcytotoxicity (CDC), and/or T cell response (e.g. CD4+ and CD8+) and/orother protective immune mechanisms.

In still additional embodiments, the invention includes a vaccinecomprising a peptide, chimeric peptide, chimeric protein, VLP, orantigenic composition as described herein and an adjuvant. In someembodiments, the adjuvant is a polymeric particle, cholera toxin, orimidazoquinoline. In further embodiments, the adjuvant formulationsinclude the classical aluminum-based adjuvants, and novel classes ofadjuvants such as liposomes (e.g., CAF01), agonists of pathogenrecognition receptors (e.g. Immune stimulating complexes (ISCOMs), LipidA analogs (MPL, RC-529, and GLA), double stranded RNA analogs (e.g. PolyI:C and Poly ICLC), cytidine monophosphate guanosineoligodeoxynucleotide (e.g. CpG, CpG ODN), flagellin, imidazoquinoline(Imiquimod and Resiquimod), polymeric particles (e.g. Chitosan),emulsions (e.g. squalene oil-based), cytokines (e.g. Interleukin-12),bacterial toxins (e.g Cholera Toxin (CT) or Escherichia coli enterotoxin(LT)), Quil A and other saponins known in the art, and the plantpolysaccharide inulin [12].

The invention also includes methods of making and methods of using anyof the foregoing compounds, compositions, articles of manufacture,apparatuses, and/or materials. Furthermore, it should be understood thataspects of the inventions that are described herein as methods canalternatively be described as “uses” of the compounds, compositions,articles, apparatuses and/or materials. All equivalent “uses” are alsocontemplated as aspects of the invention.

In some variations, the invention includes a method of producing animmune response to a Zika virus in a subject, the method comprisingadministering to the subject an effective amount of an antigeniccomposition or a vaccine as described herein, thereby producing (causingthe subject's immune system to generate) an immune response to a Zikavirus in the subject. In related variations, the disclosure provides anantigenic composition or vaccine for use in producing an immune responseto a Zika virus in a subject characterized in that producing the immuneresponse comprises administering to the subject an effective amount ofan antigenic composition or a vaccine as described herein, therebyproducing (causing the subject's immune system to generate) an immuneresponse to a Zika virus in the subject.

In some variations, the invention includes a method of treating a Zikavirus infection in a subject in need thereof, the method comprisingadministering to the subject an effective amount of an antigeniccomposition described herein, thereby treating a Zika virus infection inthe subject. In related variations, the disclosure provides an antigeniccomposition for use in treating a subject in need thereof, characterizedin that the treating comprises administering to the subject an effectiveamount of an antigenic composition described herein, thereby treating aZika virus infection in the subject.

In still additional variations, the invention includes a method ofpreventing a disease or disorder caused by a Zika virus infection in asubject, the method comprising administering to the subject an effectiveamount of an antigenic composition or a vaccine as described herein, inan amount effective to prevent a disease or disorder caused by a Zikavirus infection in the subject. In related variations, the disclosureprovides an antigenic composition or a vaccine for use in preventing adisease or disorder caused by a Zika virus infection in a subject,characterized in that the use comprises administering to the subject aneffective amount of an antigenic composition or a vaccine as describedherein, in an amount effective to prevent a disease or disorder causedby a Zika virus infection in the subject.

The invention also includes a method of protecting a subject fromdeveloping one or more symptoms of Zika virus infection, the methodcomprising administering to the subject a vaccine composition asdescribed herein, in an amount effective to protect the subject fromdeveloping one or more symptoms of Zika virus infection. In relatedvariations, the method is effective to reduce the number, severity, orduration of symptoms of a Zika virus infection. In some embodiments, thereduction in symptoms is measured as a reduction in viral load or viralcopy number in the subject. In further aspects, the disclosure providesa vaccine composition for use in protecting a subject from developingsymptoms of Zika virus infection, characterized in that the protectingcomprises administering to the subject a vaccine composition asdescribed herein, in an amount effective to protect the subject fromdeveloping symptoms of Zika virus infection.

In still another related embodiment, the invention includes a method ofimmunizing a mammalian subject against a Zika virus infection comprisingadministering to the subject an effective amount of an antigeniccomposition described herein or a vaccine described herein. In someaspects, the disclosure provides an antigenic composition or a vaccineof the disclosure for use in immunizing a mammalian subject against aZika virus infection, characterized in that the immunizing comprisesadministering to the subject an effective amount of an antigeniccomposition described herein or a vaccine described herein.

In some aspects, the disclosure provides a method of protecting asubject from sexual transmission of Zika virus, comprising administeringto the subject an effective amount of an antigenic composition orvaccine of the disclosure, thereby protecting the subject from sexualtransmission of Zika virus. In some aspects, the disclosure provides anantigenic composition or a vaccine for use in protecting a subject fromsexual transmission of Zika virus, characterized in that the protectingcomprises administering to the subject an effective amount of anantigenic composition or vaccine of the disclosure, thereby protectingthe subject from sexual transmission of Zika virus. In some embodiments,the administering is mucosal administration. In further embodiments, themucosal administration is nasal, vaginal, rectal, or oral.

The materials and methods described herein also are useful forquantifying or detecting a Zika immune response after infection and/orvaccination. For instance, the invention includes a method of detectingor measuring antibodies to Zika virus in a biological sample comprising:

-   -   a) contacting a VLP as described herein with a biological sample        under conditions suitable for the formation of an        antigen-antibody complex; and    -   b) measuring or detecting antibodies to Zika virus by detecting        or measuring an antigen-antibody complex formed between        antibodies in the biological sample and the VLP.

The invention further includes a method of detecting a Zika virusinfection comprising steps of:

-   -   a) contacting the VLP as described herein with a biological        sample from a mammalian subject under conditions suitable for        the formation of an antigen-antibody complex; and    -   b) detecting the antigen-antibody complex formed between the VLP        and antibodies in the biological sample, thereby detecting the        Zika virus infection.

In some variations, the foregoing method further comprises a step ofdetecting the Zika virus in the biological sample, wherein presence ofthe Zika virus indicates a current Zika virus infection.

The invention also includes a method for screening antibodies comprisingsteps of:

-   -   a) measuring binding of an antibody or fragment thereof to a VLP        as described herein;    -   b) measuring binding of the antibody or fragment thereof to a        Woodchuck Hepatitis core Antigen protein (WHcAg) VLP or protein;        and    -   c) determining that the antibody or fragment thereof is an        anti-Zika antibody when the antibody or fragment thereof binds        to the VLP but not the WHcAg.        Such a method is particularly useful for evaluating antibodies        produced following an immunization with VLP described herein        and/or Zika virus infection.

In any of the foregoing methods, some variations involving using the VLPin solution or suspension. In other variations, the VLP is attached to asolid support, such as any of a microbead, an assay plate, a test strip,or a filter.

The invention also includes methods of making the VLP described herein.For instance, the invention includes a method of producing a VLPcomprising introducing into a host cell the vector of claim 6 underconditions such that the cell produces the VLP. In some variations, thehost cell is a eukaryotic cell, such as a mammalian cell, a fungal oryeast cell, an insect cell, a plant cell, an amphibian cell, or an aviancell. In still other variations, the cell is a prokaryotic cell, such asa bacterial cell. An exemplary yeast host cell is a Pichia pastoris cell(e.g., Komagataella phaffii Kurtzman (ATCC® 76273™)). In somevariations, the vector is introduced into the host cell viatransformation, transfection, transduction, or electroporation. In somevariations, the cells are cultured at temperatures ranging from 25° C.to 37° C. in an incubator or fermenter or shaker, in continuousagitation and oxygenation. Optionally, the VLP produced according tosuch a method is purified from the host cell or a culture media of thehost cell. Exemplary suitable procedures for VLP purification includeprecipitation, ultracentrifugation, density gradientultracentrifugation, ultrafiltration such as tangential flow filtration(TFF) and other methods, chromatography, or a combination thereof.

The invention also includes a VLP produced by any of the foregoingmethods.

Reference throughout this specification to “one embodiment”, “someembodiments” or “an embodiment” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present disclosure. Theparticular features, structures, or characteristics described herein maybe combined in any suitable manner, and all such combinations arecontemplated as aspects of the invention.

Unless otherwise specified the use of the ordinal adjectives “first”,“second”, “third”, etc., to describe a common object, merely indicatethat different instances of like objects are being referred to, and arenot intended to imply that the objects so described must be in a givensequence, either temporally, spatially, in ranking, or in any othermanner.

The headings herein are for the convenience of the reader and notintended to be limiting. Additional aspects, embodiments, and variationsof the invention will be apparent from the Detailed Description and/orDrawing and/or claims.

Although the Applicant invented the full scope of the inventiondescribed herein, the Applicant does not intend to claim subject matterdescribed in the prior art work of others. Therefore, in the event thatstatutory prior art within the scope of a claim is brought to theattention of the Applicant by a Patent Office or other entity orindividual, the Applicant reserves the right to exercise amendmentrights under applicable patent laws to redefine the subject matter ofsuch a claim to specifically exclude such statutory prior art or obviousvariations of statutory prior art from the scope of such a claim.Variations of the invention defined by such amended claims also areintended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts elements of the WHcAg VLP system as disclosed herein forepitope delivery. The depictions on the left show a WHcAg core antigenpeptide and VLP comprised of such peptides. The images on the right showa chimeric peptide of the invention and a VLP comprised of suchpeptides. The dark black portions depict the displayed ZIKV epitope.

FIG. 2 depicts an example of a DNA construct for WHcAg chimeric VLPexpression in a yeast system.

FIGS. 3A and 3B depict the structural vaccinology strategy that wasapplied for developing WHcAg-ZIKV chimeric VLPs using the Envelopeprotein Domain III (EDIII). In FIG. 3B the EDIII sub-structural domainCD Loop is included for composition of the WHcAg-ZIKV chimeric VLP.

FIG. 4 shows a flow chart for production, purification, and qualitytesting of WHcAg-ZIKV chimerics.

FIG. 5 shows a WHcAg VLP analyzed by electron microscopy. Scale bar=50nanometers (nm).

FIG. 6 illustrates dot blot and Western blot analysis showing WHcAg-ZIKVchimeric VLP production and antigenicity. FIG. 6A demonstrates WHcAgproduction and purification form Pichia culture, WHcAg VLPs are detectedusing the commercially available monoclonal antibody HepBcAg. FIGS. 6Band 6C show WHcAg-ZIKV chimeric VLPs antigenicity using commerciallyavailable monoclonal antibodies such as ZV-2 and ZV-54 specific for ZIKVEDIII.

FIG. 7 illustrates dot blot analysis for WHcAg-ZIKV chimeric VLPsantigenicity using anti-Zika virus antibody for mouse serum, prME VLPsand ZIKV E recombinant protein are used as a positive controls for theassay.

FIG. 8 illustrates ELISA analysis of mouse serum immunized withdifferent WHcAg-ZIKV chimeric VLPs for IgG titer (A), IgG1 titer (B) andIgG2a titer (C). The limit for level of detection is 100 (dotted line).

FIG. 9 illustrates dot blot analysis of serum pools from animalsimmunized with different WHcAg-ZIKV chimeric VLPs using Zika Virus(ZIKV) Envelope (E) recombinant protein and Dengue Virus 2 (DENV-2) Erecombinant protein as antigen (FIG. 9A). Commercially availablemonoclonal antibodies (mAb) are used for assay control (FIG. 9B).

FIG. 10 shows immunofluorescence microscopy experiment demonstratingthat serum form immunized mice with WHcAg CD loop VLP vaccine candidateinduces antibodies able to recognize Zika virus in infected Vero cell inculture (left panel); the serum from the placebo control is used as anegative control in such experiment (right panel).

FIG. 11 demonstrates that WHcAg CD loop VLP vaccine candidate inducedprotective antibodies against Zika Virus in a mouse model. FIG. 11Ashows antibody dependent cell-mediated cytotoxicity (ADCC) assay: mouseserum immunized with WHcAg CD loop VLPs exert protective activity ofantibodies against Zika Virus; the serum from animals immunized withplacebo control WHcAg CTRL is included as a negative control and serumfrom an animal immunized with live Zika virus (#426) is used as anadditional control. FIG. 11B illustrates complement dependentcytotoxicity (CDC) assay: WHcAg CD loop VLPs induces CDC activity inmice immunized with such vaccine candidate in respect placebo controls(WHcAg CTRL) and an animal immunized with live Zika virus (#426).

FIG. 12 depicts an exemplary plate, test strip, and microbead of theinvention.

FIG. 13 is a depiction of a test strip of the invention and of detectionof Zika virus infection using viral epitopes expressed in VLPs using aLateral Flow Immunoassay (LFIA) system (see Example 5).

FIG. 14 shows WHcAg-ZIKV chimeric VLP Lateral Flow ImmunoassayApplication (LFIA).

FIG. 15 shows mouse models utilized for testing efficacy, safety andprotection for WHcAg-ZIKV chimera VLPs vaccine candidates.

FIG. 16 shows a mouse model utilized for testing ZIKV intrauterinetransmission protection by WHcAg-ZIKV chimera VLPs vaccine candidates.

FIG. 17 shows results of experiments analyzing serum viremia in mice 3days viral post-injection using quantitative Real-Time PCR (qRT-PCR).

DETAILED DESCRIPTION

The morphology of VLPs is pivotal for their strong immune-stimulatoryactivity: i) VLPs are more efficiently recognized by antigen presentingcells (APCs); ii) VLPs are trafficked from the site of injection to thelymph nodes; iii) the VLP structure presents a repetitive arrangement ofantigens that stimulates B-cells for the humoral immune response, andT-cells for cell mediated immune response [13, 14].

The majority of FDA approved VLP-based vaccines are currentlymanufactured in yeast due to ease of scalability. Aspects of the presentinvention are directed to a ZIKV VLP (ZIK-VLP)-based vaccine and uses ofit. In some embodiments, the VLP is produced using a yeast expressionsystem, applying structural vaccinology for the optimization of VLPimmunogenicity: antigen determinants are selectively engineered forachieving high level of immunogenicity, ZIKV specificity, and enhancedinter-strain protection [15, 16].

Terms used herein generally have the meaning that scientists in thefield would ascribe to them. The following definitions will assistunderstanding of the invention.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α-carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

“Conservative amino acid substitution” refers to the interchange of aresidue having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “nucleic acid” refers to a single or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end.

The term “encoding” refers to a polynucleotide sequence encoding one ormore amino acids. The term does not require a start or stop codon. Anamino acid sequence can be encoded in any one of six different readingframes provided by a double-stranded polynucleotide sequence. In somevariations, encoding sequences further include a start and/or a stopcodon.

A “vector” refers to a polynucleotide, which when independent of thehost chromosome, is capable of replication in a host organism. Examplesof vectors include plasmids. Vectors typically have an origin ofreplication. Vectors can comprise, e.g., transcription and translationterminators, transcription and translation initiation sequences, andpromoters useful for regulation of the expression of the particularnucleic acid.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified and that retains the modification, such as a daughter cell.Thus, for example, recombinant cells express genes that are not foundwithin the native (nonrecombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under-expressed or notexpressed at all.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. “Substantially identical”refers to two or more nucleic acids or polypeptide sequences having aspecified percentage (or specified minimum percentage) of amino acidresidues or nucleotides that are the same (i.e., (at least) 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity overa specified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the sequencecomparison algorithms below or by manual alignment and visualinspection. This definition also refers to the complement of a testsequence. Optionally, the identity or substantial identity exists over aregion that is at least about 50 nucleotides in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotidesor amino acids in length.

A “non-native amino acid” in a protein sequence refers to any amino acidother than the amino acid that occurs in the corresponding position inan alignment with a naturally-occurring polypeptide with the lowestsmallest sum probability where the comparison window is the length ofthe monomer domain queried and when compared to a naturally-occurringsequence in the non-redundant (“nr”) database of Genbank using BLAST2.0. BLAST 2.0 is described in the art [17], respectively. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (available on the world wide web atncbi.nlm.nih.gov/).

As used herein, the terms “virus-like particle” and “VLP” refer to astructure that resembles a virus. VLPs of the present disclosure lack aviral genome and are therefore noninfectious. Preferred VLPs of thepresent disclosure are derived from Woodchuck Hepatitis core Antigen(WHcAg) and thus have a VLP structure or arrangement similar to WHcAgVLPs. Virus-like particles show improved efficiency in stimulating theimmune system because they resemble the morphology of a viriondisplaying a densely repetitive array of epitopes in a limited space.Furthermore, VLPs are very safe candidates for vaccine development dueto their lack of replicating viral genetic material rendering themunable to cause viral disease. During the last decade, advancement inVLP production, purification, and adjuvant optimization has led to thelicensing of several VLP-based vaccines for the prevention of infectiousdiseases [12] such as human papilloma virus (HPV), hepatitis B virus(HBV), hepatitis E virus (HEV), and influenza. Furthermore, severalclinical trials are currently ongoing for VLP vaccines againstinfluenza, norovirus, and chikungunya virus (CHIK) (available on theworld wide web at clinicaltrials.gov/).

The term “Woodchuck Hepatitis Virus” is used interchangeably herein withthe term “Woodchuck Hepadnavirus” and refers to the virus species thatexpresses the core Antigen protein used as a platform for recombinantVLPs.

The term “chimeric” refers to a fusion of polypeptide and/or peptidessequences. “Chimeric” as used in reference to a Woodchuck Hepatitis coreAntigen (WHcAg) refers to a fusion protein of the WHcAg and an unrelatedantigen (e.g., a viral peptide and variants thereof). For instance, insome embodiments, the term “chimeric peptide” or “chimeric protein”refers to a fusion protein comprising both a WHcAg component (fulllength, or partial) and a Zika peptide or a fragment thereof. Asdescribed herein, some fusions take the form of insertions, where a Zikasequence is inserted within a WHcAg sequence.

The term “heterologous” with respect to a nucleic acid, or a polypeptidecomponent, indicates that the component occurs where it is not normallyfound in nature (e.g., relative to an adjacent component) and/or that itoriginates from a different source or species.

An “effective amount” or a “sufficient amount” of a substance is thatamount necessary to effect beneficial or desired results, includingclinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. In the context of administering anantigenic composition, an effective amount contains sufficient antigen(e.g., a VLP comprising a chimeric peptide of the disclosure) to elicitan immune response. An effective amount can be administered in one ormore doses. Efficacy can be shown in an experimental or clinical trial,for example, by comparing results achieved with a substance of interestcompared to an experimental control.

The term “dose” as used herein in reference to an antigenic compositionrefers to a measured portion of the antigenic composition taken by(administered to or received by) a subject at any one time.

The term “about” as used herein in reference to a value, encompassesfrom 90% to 110% of that value (e.g., about 200 μg VLP refers to 180 μgto 220 μg VLP).

The term “vaccination” as used herein refers to the introduction ofvaccine into a body of an organism.

A “subject” is a living multi-cellular vertebrate organism. In thecontext of this disclosure, the subject can be an experimental subject,such as a non-human mammal (e.g., a mouse, a rat, or a non-humanprimate). Alternatively, the subject can be a human subject.

An “antigenic composition” is a composition of matter suitable foradministration to a human or animal subject (e.g., in an experimental orclinical setting) that is capable of eliciting a specific immuneresponse, e.g., against a pathogen, such as Zika virus.

As such, an antigenic composition includes one or more antigens (forexample, peptide antigens) or antigenic epitopes. An antigeniccomposition can also include one or more additional components capableof eliciting or enhancing an immune response, such as an excipient,carrier, and/or adjuvant. In certain instances, antigenic compositionsare administered to elicit an immune response that protects the subjectagainst symptoms or conditions induced by a pathogen. In some cases,symptoms or disease caused by a pathogen is prevented (or reduced orameliorated) by inhibiting replication of the pathogen (e.g., virus)following exposure of the subject to the pathogen. In the context ofthis disclosure, the term antigenic composition will be understood toencompass compositions that are intended for administration to a subjector population of subjects for the purpose of eliciting a protective orpalliative immune response against a virus.

“Adjuvant” refers to a substance which, when added to a compositioncomprising an antigen, nonspecifically enhances or potentiates an immuneresponse to the antigen in the recipient upon exposure. Common adjuvantsinclude suspensions of minerals (alum, aluminum hydroxide, aluminumphosphate) onto which an antigen is adsorbed; emulsions, includingwater-in-oil, and oil-in-water (and variants thereof, including doubleemulsions and reversible emulsions), liposaccharides,lipopolysaccharides, immunostimulatory nucleic acids (such as CpGoligonucleotides), liposomes, Pattern Recognition Receptor (PRR)agonists (e.g. NALP3. RIG-I-like receptors (RIG-I and MDA5), andToll-like Receptor agonists (particularly, TLR2, TLR3, TLR4, TLR7/8 andTLR9 agonists)), and various combinations of such components [12].

An “immune response” is a response of a cell of the immune system, suchas a B cell, T cell, or monocyte, to a stimulus, such as a pathogen orantigen (e.g., formulated as an antigenic composition or a vaccine). Animmune response can be a B cell response, which results in theproduction of specific antibodies, such as antigen specific neutralizingantibodies. An immune response can also be a T cell response, such as aCD4⁺ response or a CD8⁺ response. B cell and T cell responses areaspects of a “cellular” immune response. An immune response can also bea “humoral” immune response, which is mediated by antibodies. In somecases, the response is specific for a particular antigen (that is, an“antigen-specific response”). If the antigen is derived from a pathogen,the antigen-specific response is a “pathogen-specific response.” A“protective immune response” is an immune response that inhibits adetrimental function or activity of a pathogen, reduces infection by apathogen, or decreases symptoms (including death) that result frominfection by the pathogen. A protective immune response can be measured,for example, by viral and immune assays using a serum sample from animmunized subject for testing the ability of serum antibodies forinhibition of viral replication, such as: plaque reductionneutralization test (PRNT), ELISA-neutralization assay, antibodydependent cell-mediated cytotoxicity assay (ADCC), complement-dependentcytotoxicity (CDC), antibody dependent cell-mediated phagocytosis(ADCP). In addition, vaccine efficacy can be tested by measuring the Tcell response CD4+ and CD8+ after immunization, using flow cytometry(FACS) analysis or ELISpot assay. The protective immune response can betested by measuring resistance to pathogen challenge in vivo in ananimal model. In humans, a protective immune response can bedemonstrated in a population study, comparing measurements of infection,symptoms, morbidity, mortality, etc. in treated subjects compared tountreated controls. Exposure of a subject to an immunogenic stimulus,such as a pathogen or antigen (e.g., formulated as an antigeniccomposition or vaccine), elicits a primary immune response specific forthe stimulus, that is, the exposure “primes” the immune response. Asubsequent exposure, e.g., by immunization, to the stimulus can increaseor “boost” the magnitude (or duration, or both) of the specific immuneresponse. Thus, “boosting” a preexisting immune response byadministering an antigenic composition increases the magnitude of anantigen (or pathogen) specific response, (e.g., by increasing antibodytiter and/or affinity, by increasing the frequency of antigen specific Bor T cells, by inducing maturation effector function, or a combinationthereof).

An “improved” antibody response is measured by a difference such as:protection from Zika Virus replication and viremia; neutralizingantibody titer; antibody dependent cell-mediated cytotoxicity (ADCC);complement dependent cytotoxicity (CDC), antibody dependentcell-mediated phagocytosis (ADCP); stimulation of B cell immune memory;activation of immune cells such as B cells, T cell and AntigenPresenting Cells (APC); protection from disease symptoms such as fever,pain, weight loss; weakness, maculopapular rash, Zika CongenitalSyndrome (microcephaly), Guillain-Barré Syndrome. Such differences aremeasured in a population study in which treated subjects are comparedwith untreated control subjects.

The phrase “specifically (or selectively) binds,” when referring to theinteraction between an antibody or fragment thereof and a VLP, apeptide, a chimeric protein, or a chimeric peptide as disclosed herein,refers to a binding reaction that can be determinative of the presenceof the polypeptide in a heterogeneous population of proteins (e.g., acell or tissue lysate) and other biologics. Thus, under standardconditions used in antibody binding assays, the specified VLP, peptide,or chimeric peptide binds to a particular target antibody or fragmentthereof above background (e.g., 2×, 5×, 10× or more above background)and does not bind in a significant amount to other molecules present inthe sample. Of particular interest herein are antibodies that recognizeZika virus but not Dengue virus or other flaviviruses.

As used herein, an “expression vector” is a DNA construct that containsa structural gene operably linked to an expression control sequence sothat the structural gene can be expressed when the expression vector istransformed into an appropriate host cell. Two DNA sequences are said tobe “operably linked” if the biological activity of one region willaffect the other region and also if the nature of the linkage betweenthe two DNA sequences does not (1) result in the introduction of aframe-shift mutation, (2) interfere with the ability of the promoterregion sequence to direct the transcription of the desired sequence, or(3) interfere with the ability of the desired sequence to be transcribedby the promoter region sequence. Thus, a promoter region would beoperably linked to a desired DNA sequence if the promoter were capableof effecting transcription of that desired DNA sequence. As describedherein, vectors suitable for expression in all varieties of host cellsare contemplated, including prokaryotic expression vectors andeukaryotic expression vectors. Exemplary eukaryotic expression vectorsinclude vectors for expression in mammalian cells, avian cells, insectcells, amphibian cells, plant cells, and fungal cells, including yeastcells.

Conventional or known techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology can be used to implement many elements of the invention. Suchtechniques are not always described herein in detail because they areknown and/or are explained fully in the literature, such as, MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989);Current Protocols in Molecular Biology (Ausubel et al., eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Cultureof Animal Cells: A Manual of Basic Technique (Freshney, 1987); Harlow etal., Antibodies: A Laboratory Manual (Harlow et al., 1988); and CurrentProtocols in Immunology (Coligan et al., eds., 1991).

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural reference unless the contextclearly dictates otherwise.

Zika Virus

Zika virus (ZIKV), a Flaviviridae family member, is a single-stranded,positive-sense RNA virus with an approximate 10.7 Kb genome encoding asingle polyprotein that is cleaved into three structural proteins (C,prM/M, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3,NS4A, NS4B, and NS5) by viral and host proteases [4, which isincorporated by reference herein in its entirety]. The overall structureof ZIKV soluble envelope (E) protein resembles previously reportedflavivirus E protein structures and has three distinct domains: acentral b-barrel (domain I or domain 1), an elongated finger-likestructure (domain II or domain 2), and a C-terminal immunoglobulin-likemodule (domain III or domain 3) [18]).

Chimeric Peptide Constructs

Some aspects of the invention comprise chimeric peptide or proteinconstructs having at least one portion comprised of, or derived from, arodent hepadnavirus core antigen attached to at least one portioncomprised of, or derived from, a Zika virus protein antigen. In someembodiments, the portions are joined by peptide bonds to form a chimericpolypeptide, as described below in greater detail.

A. Rodent Hepadnavirus Core Antigens

In some aspects, the chimeric hepadnavirus portion of the chimericconstruct is engineered from a rodent hepadnavirus core antigen aminoacid sequence. For instance, one or more endogenous B cell epitopes fromthe native core antigen amino acid sequence are effectively removed.Hepadnavirus core antigens are generally described in U.S. PatentApplication Publication No. 2016/0022801, which is incorporated byreference herein in its entirety.

Exemplary rodent hepadnavirus core antigens suitable for thiscomponent/portion of the chimeric construct include woodchuck (WHcAg),ground squirrel (GScAg), arctic ground squirrel (AGScAg) and human(HBcAg) hepadnavirus core antigens. An exemplary amino acid sequence ofwoodchuck hepadnavirus core antigen is set out in SEQ ID NO: 1, and isalso available as GenBank accession number NP_671816. Rodenthepadnavirus core antigens have a number of properties that make themparticularly useful for making the chimeric constructs described herein.For instance, they will self-aggregate/assemble into a multimericcomplex or VLP. The basic subunit of the core particle is a 21 kDaprotein monomer (schematically depicted in FIG. 1, top left) thatspontaneously assembles into a 240 subunit particulate structure ofabout 34 nm in diameter (FIG. 1, bottom left). The tertiary andquaternary structures of hepadnavirus core particles have beenelucidated [19, incorporated herein by reference]. The immunodominant Bcell epitope on WHcAg is localized around amino acids 76-82 of SEQ IDNO: 1 [20] forming a loop connecting adjacent alpha-helices. Thisobservation is consistent with the finding that a heterologous antigeninserted within the 76-82 loop region of HBcAg was significantly moreantigenic and immunogenic than the antigen inserted at the N- orC-termini and, importantly, more immunogenic than the antigen in thecontext of its native protein [20].

In some embodiments, the chimeric constructs of the invention arecomprised of a hepadnavirus portion that is based on a woodchuckhepadnavirus core antigen. For example, the portion used, when alignedwith SEQ ID NO:1, has an amino acid sequence that is at least 90% (e.g.,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical)to SEQ ID NO:1. The amino acid variation, relative to wildtype, can beany variation that does not destroy the self-assembling properties ofthe wildtype protein. In some variations, the variation does notincrease antigenicity of the protein, compared to wildtype. In somevariations, the changed amino acids are conservative substitutionvariants. Sequence variation can also be expressed as a limited number(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid sequence differencesbetween the wildtype sequence and the aligned sequence used in thepresent invention.

As described below, the chimeric construct preferably comprises a Zikapeptide or polypeptide sequence insert that disrupts and/or replaces theB cell epitope region of the core antigen sequence. For purposes ofsequence identity analysis in the preceding paragraphs, the changes tothe B cell epitope and the Zika insert are ignored.

B. Zika-Derived Peptides

A peptide or protein identical to or derived from a Zika virus aminoacid sequence is used in the chimeric constructs of the invention. TheZika portion has been chosen for its immunogenicity properties. Inpreferred variations, the Zika portion comprises, or is derived from, aZika Virus Envelope (E), NS1, prM, or C protein. In some variations, theZika portion comprises, or is derived from, domain 3 of a Zika Virus Eprotein. An exemplary domain 3 sequence is set forth in SEQ ID NO: 2.The use of peptides with sequence variation is contemplated, so long asthe peptide still comprises sequence that acts as an epitope that willgenerate an immune response that recognizes wildtype Zika protein orwildtype Zika virus. For instance, the peptide or protein used comprisesan amino acid sequence that is at least 90% (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2.Sequence variation can also be expressed as a limited number (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid sequence differences betweenthe wildtype sequence and the aligned sequence used in the presentinvention. In some variations, the Zika portion comprises, or is derivedfrom, NS1. An exemplary NS1 sequence is set forth in SEQ ID NO: 22. Theuse of peptides with sequence variation is contemplated, so long as thepeptide still comprises sequence that acts as an epitope that willgenerate an immune response that recognizes wildtype Zika NS1 protein orwildtype Zika virus. For instance, the peptide used comprises an aminoacid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 22. Sequencevariation can also be expressed as a limited number (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) amino acid sequence differences between thewildtype sequence and the aligned sequence used in the presentinvention. In some variations, the Zika portion comprises, or is derivedfrom, prM/M protein. An exemplary prM/M protein sequence is set forth inSEQ ID NO: 46. The use of peptides with sequence variation iscontemplated, so long as the peptide still comprises sequence that actsas an epitope that will generate an immune response that recognizeswildtype Zika prM/M protein or wildtype Zika virus. For instance, thepeptide used comprises an amino acid sequence that is at least 90%(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical) to SEQ ID NO: 46. Sequence variation can also be expressed asa limited number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acidsequence differences between the wildtype sequence and the alignedsequence used in the present invention.

In some embodiments, the peptide derived from Zika is a polypeptide offrom 4 to 200 amino acids in length. In some embodiments, the peptide isfrom 5 to 150 amino acids in length, or from 5 to 100 amino acids inlength, or from 5 to 55 amino acids in length, preferably 10 to 50 aminoacids in length, preferably 15 to 45 amino acids in length, orpreferably 20 to 40 amino acids in length. In some embodiments, thelength of the peptide is within any range having a lower limit of 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids andan independently selected upper limit of 200, 195, 190, 185, 180, 175,170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105,100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25 or 20amino acids in length, provided that the lower limit is less than theupper limit. All integer lengths from 4-200 amino acids are specificallycontemplated.

In some embodiments, the peptide derived from Zika is itself a fusionprotein comprising fragments of two, three, four or five different Zikapeptides. In various embodiments, the peptide comprises or consists ofan amino acid sequence that is at least 80% identical to a sequence asset out in any one or more of SEQ ID NOs: 2-11, 22-33, 46-47, or 50-51.In further embodiments, the peptide is 100% identical to a sequence asset out in any one or more of SEQ ID NOs: 2-11, 22-33, 46-47, or 50-51.

As described more fully below, the core antigen used herein is modifiedto include one or more Zika virus epitopes.

C. Combinatorial Technology

In some embodiments, the peptide derived from Zika is inserted into thepeptide derived from the Hepadnavirus core protein (schematicallydepicted in FIG. 1, right top) at a location that preserves theself-assembly properties of the core protein and that presents thepeptide or protein derived from Zika in an antigenic manner (FIG. 1,right bottom).

Several groups working with the HBcAg or with other VLP technologies(e.g., the L1 protein of the human papillomavirus and Qβ phage) haveopted to chemically link the foreign epitopes to the VLPs rather thaninserting the epitopes into the particles by recombinant methods. Suchembodiments are contemplated as one aspect of the invention. Thechemically conjugation approach for linking heterologous antigens hasbeen circumvented by identification of suitable insertions sites forchimeric proteins, identifiable, e.g., by combinatorial technology. (See[21]). Such techniques were used to determine 17 different insertionsites and 28 modifications of the WHcAg C-terminus that together favorassembly of chimeric particles, as well as the identification of anumber of additional improvements (see, e.g., U.S. Pat. Nos. 7,144,712;7,320,795; and 7,883,843, all incorporated herein by reference).ELISA-based screening systems have been developed that measureexpression levels, VLP assembly, and insert antigenicity using crudebacterial lysates, avoiding the need to employ labor-intensivepurification steps for VLPs that do not express and/or assemble well.

A number of insertion sites inside the loop region (positions 76-82), aswell as outside the loop region are tolerated by WHcAg. In someembodiments, the peptides or proteins are inserted directly oroptionally with linker(s) at one or both ends of the Zika peptide. Forexample, the chimeric peptides or proteins set out in SEQ ID NOs: 12-21,34-45, 48-49, and 52-53 contain portions that originate from the WHcAg(the non-underlined sequences in each of SEQ ID NOs: 12-21, 34-45,48-49, and 52-53) and portions that are the peptide derived from Zika(the underlined sequences in SEQ ID NOs: 12-21, 34-45, 48-49, and52-53).

SEQ ID NOs: 2-11 were obtained via structure analysis of Envelope (E)protein (see Examples and FIG. 3). The sequences were selected for theiradaptability with the scaffolding system, i.e., the Woodchuck Hepatitiscore Antigen (WHcAg) protein (Table 1). Specifically, SEQ ID NOs: 2-7were generated from the Envelope Domain 3 with amino acid sequence veryspecific for Zika Virus. SEQ ID NO: 8 was generated from Fusion LoopDomain that shares very similar amino acid sequence between flavivirus(e.g., Dengue Virus, Yellow Fever Virus, West Nile Virus). SEQ ID NOs: 9and 10 were generated from Envelope Domain 2 with amino acid sequencevery specific for Zika Virus. Finally, SEQ ID NO: 11 was generated fromEnvelope Domain 1 with amino acid sequence very specific for Zika Virus.

TABLE 1 SEQUENCE ID NO AMINO ACID SEQUENCE VIRUS- LIKE PARTICLE PROTEIN 1 Woodchuck MDIDPYKEFGSSYQLLNFLPLDFFPDLN HepatitisALVDTATALYEEELTGREHCSPHHTAIR Core QALVCWDELTKLIAWMSSNITSEQVRTI AntigenIVNHVNDTWGLKVRQSLWFHLSCLTFGQ (WHcAg) HTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSP RRRRSQSPRRRRSQSPSANC ZIKV E ENVELOPEANTIGEN  2 Envelope HLKCRLKMDKLRLKGVSYSLCTAAFTFT domain 3KIPAETLHGTVTVEVQYAGTDGPCKVPA full QMAVDMQTLTPVGRLITANPVITESTEN lengthSKMMLELDPPFGDSYIVIGVGEKKITHH WHRSGSTIGKAFEATVRGAKRMAV  3 EnvelopeAFTFTKIPAETLHGTVTVELQYAGTDGP domain 3 CKVPAQMAVDMQTLTPVGRLITANPVITG (EDIII) ESTENSKMMLELDPPFGDSYIVIG G loop- truncated  4 EnvelopeAFTFTKIPAETLHGTVTVELQYA domain 3, A-B loop  5 EnvelopePCKVPAQMAVDMQTLTPVGRLITANPVI domain 3, T CXCDDX loop (CD loop)  6Envelope RLITANPVITESTENSKMMLELDP domain 3, DX-E loop  7 EnvelopeGDSYIVIGVGEKKITHHWHR domain 3, F-G loop  8 Envelope DRGWGNGCGLFGK fusionloop  9 Envelope TTTVSNMAEVRSYCYEASISDMASDSRC domain 2PTQGEAYLDKQSDTQYVCKRTLVDRGWG (ED2) NGCGLFGKGSLVTCAKFACSKKMTGKSI sequenceQPENLEYR A-E 10 Envelope EASISDMASDSRCPTQGEAYLDKQSDTQ domain 2YVCKRTLVDRGWGNGCGLFGKGSLVTCA sequence KFACS B-D 11 EnvelopeMTGKSIQPENLEYRIMLSVHGSQHSGMI domain 1 VNDTGHETDENRAKVEITPNSPRAEATLglycan GGFGSLGLDCEPRTGLDFSDLYYLTM loop

Table 2 depicts chimeric peptide sequences that comprise the WoodchuckHepatitis core Antigen (WHcAg) sequence (SEQ ID NO: 1) together witheach of SEQ ID NOs: 2-11 inserted (double underline) in the region ofamino acids 77 and 82 of SEQ ID NO: 1. Amino acids in bold and italicsindicate linker sequence.

TABLE 2 WHcAg (SEQ ID NO: 1) SEQ ID PLUS SEQAMINO ACID SEQUENCE OF CHIMERIC PEPTIDE NO ID NO:WITH ZIKV ENVELOPE (E) ANTIGEN 12  2MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

HLKCRLKMDKL RLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAV

TIIVNHVND TWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQS PSANC 13  3MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNIAFTFTKIPAETLHGTVTVELQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGTIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 14  4MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNIAFTFTKIPAETLHGTVTVELQYATIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPS PRRRRSQSPRRRRSQSPSANC 15 5 MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNIPCKVPAQMAVDMQTLTPVGRLITANPVITTIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 16  6MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNIRLITANPVITESTENSKMMLELDPTIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTP SPRRRRSQSPRRRRSQSPSANC 17 7 MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNIGDSYIVIGVGEKKITHHWHRTIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRR RRSQSPRRRRSQSPSANC 18  8MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

DRGWGNGCGLFGK

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRR SQSPRRRRSQSPSANC 19  9MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

TTTVSNMAEVRSYC YEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYR

TIIVNHVNDTWG LKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSA NC 20 10MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

EASISDMASDSRCP TQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFAC S

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRR RSQSPRRRRSQSPSANC 21 11MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

MTGKSIQPENLEYR IMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTM

TIIVNHVNDTWGLKVRQSLWFH LSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC

Sequence ID NOs: 22-33 were obtained via structure analysis of NS1. Thesequences were selected for their adaptability with the scaffoldingsystem, i.e., the Woodchuck Hepatitis core Antigen (WHcAg) protein(Table 3). Structural information of the Zika Virus NS1 Protein wasobtained from published scientific literature [22].

TABLE 3 SEQ ID NO ZIKV NS1 antigen AMINO ACID SEQUENCE 22 NS1 Beta 1-2DVGCSVDFSKKETRCGT 23 NS1 Beta 3-4 DRYKYHPDSPRRLAAAVKQAWEDGICGISSVSR 24NS1 Alpha 2-Beta 5 MENIMWRSVEGELNAILEENGVQLTVVVGSV 25 NS1 Beta 4-5-6CGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNS FVVDGDTLKEC 26 NS1 IntertwinedKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNS Loop-Beta 6 FVVDG 27NS1 Beta 7-8-9 DTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYS LE 28NS1 Beta 10-11-12- CDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHL 13 IEMKTC 29NS1 Beta 12-13 GYWIESEKNDTWRLKRAHLI 30 NS1 SpaghettiRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPLS Loop-Beta 14HHNTREGYRTQMKGPWHSEELEIR 31 NS1 Beta 14-15-16-LEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEEW 17 CCRECTMPPLSFRAK 32NS1 Beta 15-16-17- CPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTM 18PPLSFRAKDGC 33 NS1 Beta 14-15-16-MKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRSTT 17-18-19-CASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEP terminus ESNLVRSMVTA

Table 4 depicts chimeric peptide sequences that comprise the WoodchuckHepatitis core Antigen (WHcAg) sequence (Sequence ID NO: 1) togetherwith each of Sequence ID NOs: 22-33 inserted (double underline) in theregion of amino acids 77 and 82 of Sequence ID NO: 1. Amino acids inbold and italics indicate linker sequence.

TABLE 4 WHcAg (SEQ. ID NO: 1) SEQ ID PLUS SEQ.AMINO ACID SEQUENCE OF CHIMERIC PROTEIN NO ID NO: WITH ZIKV NS1 ANTIGEN34 22 MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

DVGCSVDFSKKETRCGT

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSP RRRRSQSPSANC 35 23MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

DRYKYHPDSPRRLAAAV KQAWEDGICGISSVSR

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 36 24MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

MENIMWRSVEGELNAIL EENGVQLTVVVGSV

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 37 25MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

CGISSVSRMENIMWRSV EGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNSFVVDGDTLKEC

TIIVNHVNDTWGLKVRQSLWFH LSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 38 26MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

KNPMWRGPQRLPVPVNE LPHGWKAWGKSYFVRAAKTNNSFVVDG

TIIVNHVNDTWGLKVRQS LWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 39 27MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

DTLKECPLKHRAWNSFL VEDHGFGVFHTSVWLKVREDYSLE

TIIVNHVNDTWGLKVRQSLWF HLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 40 28MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

CDPAVIGTAVKGKEAVH SDLGYWIESEKNDTWRLKRAHLIEMKTC

TIIVNHVNDTWGLKVRQ SLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 41 29MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

GYWIESEKNDTWRLKRA HLI

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRS QSPRRRRSQSPSANC 42 30MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

RAHLIEMKTCEWPKSHT LWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIR

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRR RSQSPSANC 43 31MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

LEIRFEECPGTKVHVEE TCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAK

TIIVNHVN DTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 44 32MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

CPGTKVHVEETCGTRGP SLRSTTASGRVIEEWCCRECTMPPLSFRAKDGC

TIIVNHVNDTWG LKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 45 33MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

MKGPWHSEELEIRFEEC PGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVTA

TIIVNHVNDTWGLKVRQSLWFH LSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC

Sequence ID NOs: 46-47 were obtained via structure analysis of prM/Mprotein. The sequences were selected for their adaptability with thescaffolding system, i.e., the Woodchuck Hepatitis core Antigen (WHcAg)protein (Table 5). Structural information of the Zika Virus prM/Mprotein was obtained from the literature [23]. prM sequence (Sequence IDNO: 46) has been mutagenized to prevent furin protease cleavage(R89G/R90G/R92G/R93G see underlined amino acids).

TABLE 5 SEQ ID ZIKV prM/M NO antigen AMINO ACID SEQUENCE 46 prM FurinAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMC deficientDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEAGGSGGAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYS 47 M fullAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALA lengthAAAIAWLLGSSTSQKVIYLVMILLIAPAYS

Table 6 depicts chimeric peptide sequences that comprise the WoodchuckHepatitis core Antigen (WHcAg) sequence (Sequence ID NO: 1) togetherwith each of Sequence ID NOs: 46-47 inserted (double underline) in theregion of amino acids 77 and 82 of Sequence ID NO: 1. Amino acids inbold and italics indicate linker sequence.

TABLE 6 WHcAg (SEQ ID NO: 1) SEQ ID PLUS SEQ.AMINO ACID SEQUENCE OF CHIMERIC PROTEIN NO ID NO:WITH ZIKV prM/M ANTIGEN 48 46MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

AEVTRRGSAYYMYLDRN DAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEAGGSGGAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIA PAYS

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRR SQSPRRRRSQSPSANC 49 47MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

AVTLPSHSTRKLQTRSQ TWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYS

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPS PRRRRSQSPRRRRSQSPSANC

Sequence ID NOs: 50-51 were obtained via structure analysis of Capsid Cprotein. The sequences were selected for their adaptability with thescaffolding system, i.e., the Woodchuck Hepatitis core Antigen (WHcAg)protein (Table 7). Structural information of the Zika Virus Capsidprotein were obtained from the literature [24].

TABLE 7 SEQ ZIKV C ID CAPSID NO ANTIGEN AMINO ACID SEQUENCE 50 C fullMKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIR lengthMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMETIKKFKKDLAA MLRIINARKEKKRR 51C alpha 2 GHGPIRMVLAILAFLRFTAIKPSLG

Table 8 depicts chimeric peptide sequences that comprise the WoodchuckHepatitis core Antigen (WHcAg) sequence (Sequence ID NO: 1) togetherwith each of Sequence ID NOs: 50-51 inserted (double underline) in theregion of amino acids 77 and 82 of Sequence ID NO: 1. Amino acids inbold and italics indicate linker sequence.

TABLE 8 WHcAg (SEQ SEQ ID NO: 1) ID PLUS SEQAMINO ACID SEQUENCE OF CHIMERIC PROTEIN NO ID NO: WITH ZIKVC ANTIGEN 5250 MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

MKNPKKKSGGFRIVNMLKR GVARVSPFGGLKRLPAGLLLGHGPIRMVLAILAFLRFTAIKPSLGLINRWGSVGKKEAMETIKKFKKDLAAMLRIINARKEKKRR

TIIVNHVNDTW GLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 53 51MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSPHHTAIRQALVCWDELTKLIAWMSSNI

GHGPIRMVLAILAFLRFTA IKPSLG

TIIVNHVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRRGGARASRSPRRRTPSPRRRR SQSPRRRRSQSPSANC

Polynucleotides

The invention includes polynucleotides encoding the peptides as well asthe chimeric peptides described herein. Exemplary sequences are set outin SEQ ID NOs: 22-53 (Tables 3-8, respectively). Because of thedegeneracy of the genetic code, numerous polynucleotide sequences encodea given amino acid sequence, and all are contemplated as part of theinvention. In some variations, codon selection is optimized for the typeof host organism that will be used for expression.

TABLE 9 Polynucleotide sequences that encode the peptide andprotein sequences of ZIKV E antigen shown in Table 1. SEQ ID NOPOLYNUCLEOTIDE SEQUENCE VIRUS-LIKE PARTICLE PROTEIN 54 WoodchuckATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCA HepatitisATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGA CoreACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAA AntigenTTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTAT (WHcAg)CAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCACTTCTGAACAAGTTAGAACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT ZIKV E ENVELOPE ANTIGEN 55 EnvelopeCACTTGAAGTGTAGATTGAAGATGGACAAGTTGAGATTGAA domainGGGTGTTTCTTACTCTTTGTGTACTGCTGCTTTCACTTTCA 3 fullCTAAGATCCCAGCTGAAACTTTGCACGGTACTGTTACTGTT lengthGAAGTTCAATACGCTGGTACTGACGGTCCATGTAAGGTTCCAGCTCAAATGGCTGTTGACATGCAAACTTTGACTCCAGTTGGTAGATTGATCACTGCTAACCCAGTTATCACTGAATCTACTGAAAACTCTAAGATGATGTTGGAATTGGACCCACCATTCGGTGACTCTTACATCGTTATCGGTGTTGGTGAAAAGAAGATCACTCACCACTGGCACAGATCTGGTTCTACTATCGGTAAGGCTTTCGAAGCTACTGTTAGAGGTGCTAAGAGAATGGCTGTT 56 EnvelopeGCTTTCACTTTCACTAAGATCCCAGCTGAAACTTTGCACGG domain 3TACTGTTACTGTTGAATTGCAATACGCTGGTACTGACGGTC (EDIII)CATGTAAGGTTCCAGCTCAAATGGCTGTTGACATGCAAACT G loop-TTGACTCCAGTTGGTAGATTGATCACTGCTAACCCAGTTAT truncatedCACTGAATCTACTGAAAACTCTAAGATGATGTTGGAATTGGACCCACCATTCGGTGACTCTTACATCGTTATCGGT 57 EnvelopeGCTTTCACTTTCACTAAGATCCCAGCTGAAACTTTGCACGG domainTACTGTTACTGTTGAATTGCAATACGCT 3, A-B loop 58 EnvelopeCCATGTAAGGTTCCAGCTCAAATGGCTGTTGACATGCAAA domainCTTTGACTCCAGTTGGTAGATTGATCACTGCTAACCCAGT 3, TATCACT CXCDDX loop (CDloop) 59 Envelope AGATTGATCACTGCTAACCCAGTTATCACTGAATCTACT domainGAAAACTCTAAGATGATGTTGGAATTGGACCCA 3, DX-E loop 60 EnvelopeGGTGACTCTTACATCGTTATCGGTGTTGGTGAAAAGAAGAT domain CACTCACCACTGGCACAGA3, F-G loop 61 Envelope GACAGAGGTTGGGGTAACGGTTGTGGTTTGTTCGGTAAG fusionloop VIRUS-LIKE PARTICLE PROTEIN 54 WoodchuckATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCA HepatitisATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGA CoreACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAA AntigenTTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTAT (WHcAg)CAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCACTTCTGAACAAGTTAGAACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT ZIKV E ENVELOPE ANTIGEN 62 EnvelopeACTACTACTGTTTCTAACATGGCTGAAGTTAGATCTT domainACTGTTACGAAGCTTCTATCTCTGACATGGCTTCTGA 2 (ED2)CTCTAGATGTCCAACTCAAGGTGAAGCTTACTTGGAC sequenceAAGCAATCTGACACTCAATACGTTTGTAAGAGAACTT A-ETGGTTGACAGAGGTTGGGGTAACGGTTGTGGTTTGTTCGGTAAGGGTTCTTTGGTTACTTGTGCTAAGTTCGCTTGTTCTAAGAAGATGACTGGTAAGTCTATCCAACCAG AAAACTTGGAATACAGA 63 EnvelopeGAAGCTTCTATCTCTGACATGGCTTCTGACTCTAGAT domain 2GTCCAACTCAAGGTGAAGCTTACTTGGACAAGCAATC sequenceTGACACTCAATACGTTTGTAAGAGAACTTTGGTTGAC B-DAGAGGTTGGGGTAACGGTTGTGGTTTGTTCGGTAAGGGTTCTTTGGTTACTTGTGCTAAGTTCGCTTGTTCT 64 EnvelopeATGACTGGTAAGTCTATCCAACCAGAAAACTTGGAATACAG domain 1AATCATGTTGTCTGTTCACGGTTCTCAACACTCTGGTATGA glycanTCGTTAACGACACTGGTCACGAAACTGACGAAAACAGAGCT loopAAGGTTGAAATCACTCCAAACTCTCCAAGAGCTGAAGCTACTTTGGGTGGTTTCGGTTCTTTGGGTTTGGACTGTGAACCAAGAACTGGTTTGGACTTCTCTGACTTGTACTACTTGACTATG

TABLE 10 Polynucleotide sequences encoding WHcAg-ZIKV chimeric proteinswith ZIKV E antigen shown in Table 2. WHcAg POLYNUCLEOTIDE SEQSEQUENCE (SEQ ID ID NO: 54) PLUS POLYNUCLEOTIDE SEQUENCE OF CHIMERIC NOSEQ ID NO: PROTEIN WITH ZIKV E ANTIGEN 65 55ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTCACTTGAAGTGTAGATTGAAGATGGACAAGTTGAGATTGAAGGGTGTTTCTTACTCTTTGTGTACTGCTGCTTTCACTTTCACTAAGATCCCAGCTGAAACTTTGCACGGTACTGTTACTGTTGAAGTTCAATACGCTGGTACTGACGGTCCATGTAAGGTTCCAGCTCAAATGGCTGTTGACATGCAAACTTTGACTCCAGTTGGTAGATTGATCACTGCTAACCCAGTTATCACTGAATCTACTGAAAACTCTAAGATGATGTTGGAATTGGACCCACCATTCGGTGACTCTTACATCGTTATCGGTGTTGGTGAAAAGAAGATCACTCACCACTGGCACAGATCTGGTTCTACTATCGGTAAGGCTTTCGAAGCTACTGTTAGAGGTGCTAAGAGAATGGCTGTTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 66 56ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGCTTTCACTTTCACTAAGATCCCAGCTGAAACTTTGCACGGTACTGTTACTGTTGAATTGCAATACGCTGGTACTGACGGTCCATGTAAGGTTCCAGCTCAAATGGCTGTTGACATGCAAACTTTGACTCCAGTTGGTAGATTGATCACTGCTAACCCAGTTATCACTGAATCTACTGAAAACTCTAAGATGATGTTGGAATTGGACCCACCATTCGGTGACTCTTACATCGTTATCGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGC TAACTGT 67 57ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGCTTTCACTTTCACTAAGATCCCAGCTGAAACTTTGCACGGTACTGTTACTGTTGAATTGCAATACGCTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 68 58ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCCCATGTAAGGTTCCAGCTCAAATGGCTGTTGACATGCAAACTTTGACTCCAGTTGGTAGATTGATCACTGCTAACCCAGTTATCACTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAA TCTCCATCTGCTAACTGT 69 59ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCAGATTGATCACTGCTAACCCAGTTATCACTGAATCTACTGAAAACTCTAAGATGATGTTGGAATTGGACCCAACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAAC TGT 70 60ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGACTCTTACATCGTTATCGGTGTTGGTGAAAAGAAGATCACTCACCACTGGCACAGAACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 71 61ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTACTGACAGAGGTTGGGGTAACGGTTGTGGTTTGTTCGGTAAGGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAA GATCTCAATCTCCATCTGCTAACTGT 7262 ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTACTACTACTGTTTCTAACATGGCTGAAGTTAGATCTTACTGTTACGAAGCTTCTATCTCTGACATGGCTTCTGACTCTAGATGTCCAACTCAAGGTGAAGCTTACTTGGACAAGCAATCTGACACTCAATACGTTTGTAAGAGAACTTTGGTTGACAGAGGTTGGGGTAACGGTTGTGGTTTGTTCGGTAAGGGTTCTTTGGTTACTTGTGCTAAGTTCGCTTGTTCTAAGAAGATGACTGGTAAGTCTATCCAACCAGAAAACTTGGAATACAGAGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGA TCTCAATCTCCATCTGCTAACTGT73 63 ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGAAGCTTCTATCTCTGACATGGCTTCTGACTCTAGATGTCCAACTCAAGGTGAAGCTTACTTGGACAAGCAATCTGACACTCAATACGTTTGTAAGAGAACTTTGGTTGACAGAGGTTGGGGTAACGGTTGTGGTTTGTTCGGTAAGGGTTCTTTGGTTACTTGTGCTAAGTTCGCTTGTTCTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAAC TGT 74 64ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTATGACTGGTAAGTCTATCCAACCAGAAAACTTGGAATACAGAATCATGTTGTCTGTTCACGGTTCTCAACACTCTGGTATGATCGTTAACGACACTGGTCACGAAACTGACGAAAACAGAGCTAAGGTTGAAATCACTCCAAACTCTCCAAGAGCTGAAGCTACTTTGGGTGGTTTCGGTTCTTTGGGTTTGGACTGTGAACCAAGAACTGGTTTGGACTTCTCTGACTTGTACTACTTGACTATGGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAG ATCTCAATCTCCATCTGCTAACTGT

TABLE 11 Polynucleotide sequences that encode the peptide and proteinsequences of ZIKV NS1 antigen shown in Table 3. SEQ ZIKV NS1 ID NOANTIGEN POLYNUCLEOTIDE SEQUENCE OF ZIKV NS1 ANTIGEN 75 NS1 Beta 1-GACGTTGGTTGTTCTGTTGACTTCTCTAAGAAGGAAACTAGATGTGGTAC 2 T 76 NS1 Beta 3-GACAGATACAAGTACCACCCAGACTCTCCAAGAAGATTGGCTGCTGCTGT 4TAAGCAAGCTTGGGAAGACGGTATCTGTGGTATCTCTTCTGTTTCTAGA 77 NS1 AlphaATGGAAAACATCATGTGGAGATCTGTTGAAGGTGAATTGAACGCTATCTT 2-Beta 5GGAAGAAAACGGTGTTCAATTGACTGTTGTTGTTGGTTCTGTT 78 NS1 Beta 4-TGTGGTATCTCTTCTGTTTCTAGAATGGAAAACATCATGTGGAGATCTGT 5-6TGAAGGTGAATTGAACGCTATCTTGGAAGAAAACGGTGTTCAATTGACTGTTGTTGTTGGTTCTGTTAAGAACCCAATGTGGAGAGGTCCACAAAGATTGCCAGTTCCAGTTAACGAATTGCCACACGGTTGGAAGGCTTGGGGTAAGTCTTACTTCGTTAGAGCTGCTAAGACTAACAACTCTTTCGTTGTTGACGGTG ACACTTTGAAGGAATGTGTT79 NS1 Inter. AAGAACCCAATGTGGAGAGGTCCACAAAGATTGCCAGTTCCAGTTAACGALoop-Beta 6 ATTGCCACACGGTTGGAAGGCTTGGGGTAAGTCTTACTTCGTTAGAGCTGCTAAGACTAACAACTCTTTCGTTGTTGACGGT 80 NS1 Beta 7-GACACTTTGAAGGAATGTCCATTGAAGCACAGAGCTTGGAACTCTTTCTT 8-9GGTTGAAGACCACGGTTTCGGTGTTTTCCACACTTCTGTTTGGTTGAAGGTTAGAGAAGACTACTCTTTGGAA 81 NS1 BetaTGTGACCCAGCTGTTATCGGTACTGCTGTTAAGGGTAAGGAAGCTGTTCA 10-11-12-13CTCTGACTTGGGTTACTGGATCGAATCTGAAAAGAACGACACTTGGAGATTGAAGAGAGCTCACTTGATCGAAATGAAGACTTGT 82 NS1 BetaGGTTACTGGATCGAATCTGAAAAGAACGACACTTGGAGATTGAAGAGAGC 12-13 TCACTTGATC 83NS1 AGAGCTCACTTGATCGAAATGAAGACTTGTGAATGGCCAAAGTCTCACAC SpaghettiTTTGTGGACTGACGGTATCGAAGAATCTGACTTGATCATCCCAAAGTCTT Loop-BetaTGGCTGGTCCATTGTCTCACCACAACACTAGAGAAGGTTACAGAACTCAA 14ATGAAGGGTCCATGGCACTCTGAAGAATTGGAAATCAGA 84 NS1 BetaTTGGAAATCAGATTCGAAGAATGTCCAGGTACTAAGGTTCACGTTGAAGA 14-15-16-17AACTTGTGGTACTAGAGGTCCATCTTTGAGATCTACTACTGCTTCTGGTAGAGTTATCGAAGAATGGTGTTGTAGAGAATGTACTATGCCACCATTGTCT TTCAGAGCTAAG 85NS1 Beta TGTCCAGGTACTAAGGTTCACGTTGAAGAAACTTGTGGTACTAGAGGTCC 15-16-17-18ATCTTTGAGATCTACTACTGCTTCTGGTAGAGTTATCGAAGAATGGTGTTGTAGAGAATGTACTATGCCACCATTGTCTTTCAGAGCTAAGGACGGTTGT 86 NS1 BetaATGAAGGGTCCATGGCACTCTGAAGAATTGGAAATCAGATTCGAAGAATG 14-15-16-TCCAGGTACTAAGGTTCACGTTGAAGAAACTTGTGGTACTAGAGGTCCAT 17-18-19-C-CTTTGAGATCTACTACTGCTTCTGGTAGAGTTATCGAAGAATGGTGTTGT term.AGAGAATGTACTATGCCACCATTGTCTTTCAGAGCTAAGGACGGTTGTTGGTACGGTATGGAAATCAGACCAAGAAAGGAACCAGAATCTAACTTGGTTA GATCTATGGTTACTGCT

TABLE 12 Polynucleotide sequences encoding WHcAg-ZIKV chimericproteins with ZIKV NS1 antigen shown in Table 4. WHcAg (SEQ ID SEQNO: 54) ID PLUS SEQ POLYNUCLEOTIDE SEQUENCE OF CHIMERIC PROTEIN WITH NOID NO: ZIKV NS1 ANTIGEN 87 75ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTGACGTTGGTTGTTCTGTTGACTTCTCTAAGAAGGAAACTAGATGTGGTACTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCA TCTGCTAACTGT 88 76ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTGACAGATACAAGTACCACCCAGACTCTCCAAGAAGATTGGCTGCTGCTGTTAAGCAAGCTTGGGAAGACGGTATCTGTGGTATCTCTTCTGTTTCTAGAGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCT GCTAACTGT 89 77ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTATGGAAAACATCATGTGGAGATCTGTTGAAGGTGAATTGAACGCTATCTTGGAAGAAAACGGTGTTCAATTGACTGTTGTTGTTGGTTCTGTTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAAC TGT 90 78ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTTGTGGTATCTCTTCTGTTTCTAGAATGGAAAACATCATGTGGAGATCTGTTGAAGGTGAATTGAACGCTATCTTGGAAGAAAACGGTGTTCAATTGACTGTTGTTGTTGGTTCTGTTAAGAACCCAATGTGGAGAGGTCCACAAAGATTGCCAGTTCCAGTTAACGAATTGCCACACGGTTGGAAGGCTTGGGGTAAGTCTTACTTCGTTAGAGCTGCTAAGACTAACAACTCTTTCGTTGTTGACGGTGACACTTTGAAGGAATGTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 91 79ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTAAGAACCCAATGTGGAGAGGTCCACAAAGATTGCCAGTTCCAGTTAACGAATTGCCACACGGTTGGAAGGCTTGGGGTAAGTCTTACTTCGTTAGAGCTGCTAAGACTAACAACTCTTTCGTTGTTGACGGTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 92 80ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTGACACTTTGAAGGAATGTCCATTGAAGCACAGAGCTTGGAACTCTTTCTTGGTTGAAGACCACGGTTTCGGTGTTTTCCACACTTCTGTTTGGTTGAAGGTTAGAGAAGACTACTCTTTGGAAGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 93 81ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTTGTGACCCAGCTGTTATCGGTACTGCTGTTAAGGGTAAGGAAGCTGTTCACTCTGACTTGGGTTACTGGATCGAATCTGAAAAGAACGACACTTGGAGATTGAAGAGAGCTCACTTGATCGAAATGAAGACTTGTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 94 82ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTGGTTACTGGATCGAATCTGAAAAGAACGACACTTGGAGATTGAAGAGAGCTCACTTGATCGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 95 83ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTAGAGCTCACTTGATCGAAATGAAGACTTGTGAATGGCCAAAGTCTCACACTTTGTGGACTGACGGTATCGAAGAATCTGACTTGATCATCCCAAAGTCTTTGGCTGGTCCATTGTCTCACCACAACACTAGAGAAGGTTACAGAACTCAAATGAAGGGTCCATGGCACTCTGAAGAATTGGAAATCAGAGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 96 84ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTTTGGAAATCAGATTCGAAGAATGTCCAGGTACTAAGGTTCACGTTGAAGAAACTTGTGGTACTAGAGGTCCATCTTTGAGATCTACTACTGCTTCTGGTAGAGTTATCGAAGAATGGTGTTGTAGAGAATGTACTATGCCACCATTGTCTTTCAGAGCTAAGGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 97 85ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTTGTCCAGGTACTAAGGTTCACGTTGAAGAAACTTGTGGTACTAGAGGTCCATCTTTGAGATCTACTACTGCTTCTGGTAGAGTTATCGAAGAATGGTGTTGTAGAGAATGTACTATGCCACCATTGTCTTTCAGAGCTAAGGACGGTTGTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCT GCTAACTGT 98 86ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTATGAAGGGTCCATGGCACTCTGAAGAATTGGAAATCAGATTCGAAGAATGTCCAGGTACTAAGGTTCACGTTGAAGAAACTTGTGGTACTAGAGGTCCATCTTTGAGATCTACTACTGCTTCTGGTAGAGTTATCGAAGAATGGTGTTGTAGAGAATGTACTATGCCACCATTGTCTTTCAGAGCTAAGGACGGTTGTTGGTACGGTATGGAAATCAGACCAAGAAAGGAACCAGAATCTAACTTGGTTAGATCTATGGTTACTGCTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT

TABLE 13 Polynucleotide sequences that encode the peptide and protein sequences of ZIKV prM/M antigen shown in Table 5. SEQ ZIKV ID prM/M NOANTIGEN POLYNUCLEOTIDE SEQUENCE OF ZIKV prM/M ANTIGEN  99 prM FurinGCTGAAGTTACTAGAAGAGGTTCTGCTTACTACATGTACTTGGACAGAAACG deficientACGCTGGTGAAGCTATCTCTTTCCCAACTACTTTGGGTATGAACAAGTGTTACATCCAAATCATGGACTTGGGTCACATGTGTGACGCTACTATGTCTTACGAATGTCCAATGTTGGACGAAGGTGTTGAACCAGACGACGTTGACTGTTGGTGTAACACTACTTCTACTTGGGTTGTTTACGGTACTTGTCACCACAAGAAGGGTGAAGCTGGTGGTTCTGGTGGTGCTGTTACTTTGCCATCTCACTCTACTAGAAAGTTGCAAACTAGATCTCAAACTTGGTTGGAATCTAGAGAATACACTAAGCACTTGATCAGAGTTGAAAACTGGATCTTCAGAAACCCAGGTTTCGCTTTGGCTGCTGCTGCTATCGCTTGGTTGTTGGGTTCTTCTACTTCTCAAAAGGTTATCTACTTGGTTATGATCTTGTTGATCGCTCCAGCTTACTCT 100 M fullGCTGTTACTTTGCCATCTCACTCTACTAGAAAGTTGCAAACTAGATCTCAAA lengthCTTGGTTGGAATCTAGAGAATACACTAAGCACTTGATCAGAGTTGAAAACTGGATCTTCAGAAACCCAGGTTTCGCTTTGGCTGCTGCTGCTATCGCTTGGTTGTTGGGTTCTTCTACTTCTCAAAAGGTTATCTACTTGGTTATGATCTTGTTGA TCGCTCCAGCTTACTCT

TABLE 14 Polynucleotide sequences encoding WHcAg-ZIKV chimericproteins with ZIKV prM/M antigen shown in Table 6. WHcAg (SEQ ID SEQNO: 62) ID PLUS SEQ POLYNUCLEOTIDE SEQUENCE OF CHIMERIC PROTEIN WITH NOID NO: ZIKV prM/M ANTIGEN 101  99ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTGCTGAAGTTACTAGAAGAGGTTCTGCTTACTACATGTACTTGGACAGAAACGACGCTGGTGAAGCTATCTCTTTCCCAACTACTTTGGGTATGAACAAGTGTTACATCCAAATCATGGACTTGGGTCACATGTGTGACGCTACTATGTCTTACGAATGTCCAATGTTGGACGAAGGTGTTGAACCAGACGACGTTGACTGTTGGTGTAACACTACTTCTACTTGGGTTGTTTACGGTACTTGTCACCACAAGAAGGGTGAAGCTGGTGGTTCTGGTGGTGCTGTTACTTTGCCATCTCACTCTACTAGAAAGTTGCAAACTAGATCTCAAACTTGGTTGGAATCTAGAGAATACACTAAGCACTTGATCAGAGTTGAAAACTGGATCTTCAGAAACCCAGGTTTCGCTTTGGCTGCTGCTGCTATCGCTTGGTTGTTGGGTTCTTCTACTTCTCAAAAGGTTATCTACTTGGTTATGATCTTGTTGATCGCTCCAGCTTACTCTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCT AACTGT 102 100ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTGCTGTTACTTTGCCATCTCACTCTACTAGAAAGTTGCAAACTAGATCTCAAACTTGGTTGGAATCTAGAGAATACACTAAGCACTTGATCAGAGTTGAAAACTGGATCTTCAGAAACCCAGGTTTCGCTTTGGCTGCTGCTGCTATCGCTTGGTTGTTGGGTTCTTCTACTTCTCAAAAGGTTATCTACTTGGTTATGATCTTGTTGATCGCTCCAGCTTACTCTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT

TABLE 15 Polynucleotide sequences that encode the peptide and proteinsequences of ZIKV Capsid C antigen shown in Table 7. SEQ ZIKV C IDCAPSID NO ANTIGEN POLYNUCLEOTIDE SEQUENCE OF ZIKV C ANTIGEN 103 C fullATGAAGAACCCAAAGAAGAAGTCTGGTGGTTTCAGAATCGTTAACATGTT lengthGAAGAGAGGTGTTGCTAGAGTTTCTCCATTCGGTGGTTTGAAGAGATTGCCAGCTGGTTTGTTGTTGGGTCACGGTCCAATCAGAATGGTTTTGGCTATCTTGGCTTTCTTGAGATTCACTGCTATCAAGCCATCTTTGGGTTTGATCAACAGATGGGGTTCTGTTGGTAAGAAGGAAGCTATGGAAACTATCAAGAAGTTCAAGAAGGACTTGGCTGCTATGTTGAGAATCATCAACGCTAGAAAGGAA AAGAAGAGAAGA 104C alpha 2 GGTCACGGTCCAATCAGAATGGTTTTGGCTATCTTGGCTTTCTTGAGATTCACTGCTATCAAGCCATCTTTGGGT

TABLE 16 Polynucleotide sequences encoding WHcAg-ZIKV chimericproteins with ZIKV C antigen shown in Table 8. WHcAg (SEQ ID SEQ NO: 62)ID PLUS SEQ POLYNUCLEOTIDE SEQUENCE OF CHIMERIC PROTEIN WITH NO ID NO:ZIKV C ANTIGEN 105 103ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTATGAAGAACCCAAAGAAGAAGTCTGGTGGTTTCAGAATCGTTAACATGTTGAAGAGAGGTGTTGCTAGAGTTTCTCCATTCGGTGGTTTGAAGAGATTGCCAGCTGGTTTGTTGTTGGGTCACGGTCCAATCAGAATGGTTTTGGCTATCTTGGCTTTCTTGAGATTCACTGCTATCAAGCCATCTTTGGGTTTGATCAACAGATGGGGTTCTGTTGGTAAGAAGGAAGCTATGGAAACTATCAAGAAGTTCAAGAAGGACTTGGCTGCTATGTTGAGAATCATCAACGCTAGAAAGGAAAAGAAGAGAAGAGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT 106 104ATGGACATCGACCCATACAAGGAATTCGGTTCTTCTTACCAATTGTTGAACTTCTTGCCATTGGACTTCTTCCCAGACTTGAACGCTTTGGTTGACACTGCTACTGCTTTGTACGAAGAAGAATTGACTGGTAGAGAACACTGTTCTCCACACCACACTGCTATCAGACAAGCTTTGGTTTGTTGGGACGAATTGACTAAGTTGATCGCTTGGATGTCTTCTAACATCGGTGGTGGTGGTACTGGTCACGGTCCAATCAGAATGGTTTTGGCTATCTTGGCTTTCTTGAGATTCACTGCTATCAAGCCATCTTTGGGTGGTGGTGGTGGTACTATCATCGTTAACCACGTTAACGACACTTGGGGTTTGAAGGTTAGACAATCTTTGTGGTTCCACTTGTCTTGTTTGACTTTCGGTCAACACACTGTTCAAGAATTCTTGGTTTCTTTCGGTGTTTGGATCAGAACTCCAGCTCCATACAGACCACCAAACGCTCCAATCTTGTCTACTTTGCCAGAACACACTGTTATCAGAAGAAGAGGTGGTGCTAGAGCTTCTAGATCTCCAAGAAGAAGAACTCCATCTCCAAGAAGAAGAAGATCTCAATCTCCAAGAAGAAGAAGATCTCAATCTCCATCTGCTAACTGT

Methods of making polynucleotides of a predetermined sequence arewell-known. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides andAnalogues, 1st Ed. (Oxford University Press, New York, 1991).Solid-phase synthesis methods are preferred for both polyribonucleotidesand polydeoxyribonucleotides (the well-known methods of synthesizing DNAare also useful for synthesizing RNA). Polyribonucleotides can also beprepared enzymatically. Non-naturally occurring nucleobases can beincorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No.7,223,833; [25, 26].

Vectors

In some embodiments, vectors are used to express the polynucleotidesdescribed herein. Expression vectors generally include expressioncontrol sequences selected for a type of host cell to be used forprotein expression. In some embodiments, the expression vector is ayeast expression vector. Various expression vectors are known in theart, including but not limited to pD912 or pD902 for secretory orcytosolic production of VLPs respectively (ATUM (available on the worldwide web at atum.bio/)). Components and structure of an exemplaryexpression vector is depicted in FIG. 2.

VLP Production/Purification

A number of appropriate yeast strains for protein expression exist,including but not limited to Komagataella phaffii Kurtzman (ATCC®76273™) or Komagataella pastoris (ATCC® 76274™). Using in silicoanalysis we have designed codon optimized DNA constructs expressing theZika Virus antigens conserved between different strains (FIGS. 3A and3B). VLPs are produced by recombinant constructs using the promoter fromthe Pichia alcohol oxidase 1 (AOX1) gene to drive production of therecombinant protein according to ATUM (available on the world wide webat atum.bio/) with further optimization. VLPs are purified by, forexample, precipitation, ultracentrifugation, chromatography, tangentialflow filtration (TFF) or ultrafiltration methods or combination of suchmethods (FIG. 4) [16]. VLPs are quantified for purity and antigenicityusing biochemical and immune assays such as Western blotting, dot blot,ELISA (FIGS. 6 and 7), gel electrophoresis (SDS-PAGE or native Agarosegel, combined with Coomassie Blue staining), and electron microscopy(FIG. 5) [16].

Antigenic and Immunogenic Characterization of VLPs

A. Antigenicity

Prior to immunogenicity testing, VLPs comprising a chimeric peptide asdescribed herein are characterized for expression, particle assembly,and ability to bind a peptide-specific antibody. Capture enzyme-linkedimmunosorbent assays (ELISAs), dot blot or Western blot are utilized anddesigned to assess three VLP properties according to methods known inthe art [16](FIGS. 6 and 7): 1) protein expression of the WHcAgpolypeptide by use of an antibody that is specific for the WHcAg (e.g.Santa Cruz Biotechnology, antibody Hep B cAg Antibody (13A9): sc-23946);2) particle assembly using an antibody specific for a conformationalepitope on WHcAg; and 3) display of the epitope of a Zika peptide of thedisclosure by use of Zika peptide-reactive antibodies (e.g., ATCC BEIResources NR-50414 Monoclonal Anti-Zika Virus Envelope (E) Protein,Clone ZV-2). Constructs that are positive for all three properties areselected for further purification (e.g., Ultracentrifugation,Ultrafiltration, Chromatography). In brief, expression, particleassembly, and antibody binding are assayed by ELISA, dot blot, andWestern blotting. SDS-PAGE and Agarose electrophoresis, along withelectron microscopy (FIG. 5), are used to assess the purity and assemblyof VLPs. VLPs can be tested for non-cross-reactivity using in vitroAntibody-Dependent Enhancement Assay according to the literature [27].

B. Immunogenicity

VLP-based vaccine antigenicity is assessed in different adjuvantformulations in animal model such as immunocompetent mouse model (e.g.BALB/c) or immunodeficient mouse model (e.g. AG129 and A129). The immuneresponse to VLPs is assessed in mice models for Zika Virus infectionaccording to the literature [27, 28]. In addition to anti-insert,anti-peptide-protein and anti-WHcAg antibody endpoint titers, antibodyspecificity, isotype distribution, antibody persistence and antibodyavidity are monitored. VLPs immune stimulation can be tested forinducing non-cross-reactivity antibody analyzing serum samples of VLPimmunized mice by dot blot analysis (FIG. 9) or in vitro forAntibody-Dependent Enhancement Assay according to the literature [27].Immune sera are compared to the activity of a reference antibody byELISA and neutralization assays known in the art [16, 28]. Immuneresponses are tested in vivo in various mammalian species (e.g., rodentssuch as rats and mice, nonhuman primates (NHP), and/or humans).

Compositions

The invention includes compositions that comprise a chimeric peptide orVLP described herein or a polynucleotide encoding the chimeric peptide.In some embodiments, the composition is an antigenic composition. Insome embodiments, the composition further comprises a pharmaceuticallyacceptable carrier. The term “carrier” refers to a vehicle within whichthe VLP, vector, chimeric peptide or polynucleotide encoding thechimeric peptide is administered to a mammalian subject. The termcarrier encompasses diluents, excipients, adjuvants and combinationsthereof. Pharmaceutically acceptable carriers are well known in the art(see, e.g., Remington's Pharmaceutical Sciences by Martin, 1975).

Exemplary “diluents” include sterile liquids such as sterile water,saline solutions, and buffers (e.g., phosphate, tris, borate, succinate,or histidine). Exemplary “excipients” are inert substances that mayenhance vaccine stability and include but are not limited to polymers(e.g., polyethylene glycol), carbohydrates (e.g., starch, glucose,lactose, sucrose, or cellulose), and alcohols (e.g., glycerol, sorbitol,or xylitol).

Adjuvants are broadly separated into two classes based upon theirprimary mechanism of action: vaccine delivery systems (e.g., emulsions,microparticles, immune stimulating complexes (ISCOMS), or liposomes)that target associated antigens to antigen presenting cells (APC); andimmunostimulatory adjuvants (e.g., LPS, MPL, or CpG) that directlyactivate innate immune responses. Different types of adjuvants can becombined to enhance their immunostimulatory activity (e.g. AS04 (GSK) iscomposed of MPL mixed with an aluminum salt).

A. Traditional and Molecular Adjuvants

Although adjuvants are not required when using the WHcAg delivery systemdisclosed herein, some embodiments of the present invention employadjuvant formulations. Adjuvants are a class of immunomodulatorymolecules and compositions able to augment vaccine effectiveness andsafety by: 1) enhancing immunogenicity and increasing the duration ofprotection; 2) broadening the induction of the immune response; 3)reducing vaccine dosage and vaccination cost (antigen sparing); 4)accelerating the immune response; 5) stimulating a strongerimmunological memory; 6) improving efficacy in weak responder patientssuch as neonates, the elderly and immunocompromised individuals [12]. Inaddition, some adjuvants formulation may also increase VLP-based vaccinestability and play an important role in VLPs delivery. Adjuvantformulations for this disclosure includes the classical aluminum-basedadjuvants, and novel classes of adjuvants such as liposomes (e.g.,CAF01), agonists of pathogen recognition receptors (e.g. Immunestimulating complexes (ISCOMs), Lipid A analogs (MPL, RC-529, and GLA),double stranded RNA analogs (e.g. Poly I:C and Poly ICLC), cytidinemonophosphate guanosine oligodeoxynucleotide (e.g. CpG, CpG ODN),flagellin, imidazoquinoline (Imiquimod and Resiquimod), polymericparticles (e.g. Chitosan), emulsions (e.g. squalene oil-based),cytokines (e.g. Interleukin-12), bacterial toxins (e.g Cholera Toxin(CT) or Escherichia coli enterotoxin (LT)), Quil A and other saponinsknown in the art, and the plant polysaccharide inulin [12].Specifically, immunization in saline effectively elicits immune responseagainst the vaccine preparation antigen/s. However, formulation innon-inflammatory agents such as IFA (mineral oil), Montanide ISA 720(squalene), and aluminum phosphate (AlP04), or immunomodulatory agentsor adjuvants enhance vaccine immunogenicity. Additionally,administration of WHcAg results in the production of multiple IgGisotypes, regardless of which if any adjuvant is employed. The WHcAgVLPs have shown superior stability as compared to recombinant proteinfrom subunit vaccines in the particularly harsh mucosal environment.This characteristic is quite advantageous for developing vaccines formucosal administration such as the oral, nasal, rectal and vaginalroute. Inclusion of a CpG motif also enhances the primary response.Moreover, use of an inflammatory adjuvant such as the Ribi formulationis not more beneficial than is the use of non-inflammatory adjuvants,indicating that the benefits of the adjuvants result from a depot effectrather than from non-specific inflammation. Thus, the core platform isused with no adjuvant or with non-inflammatory adjuvants depending uponthe application and the quantity of antibody desired. In someembodiments of the present disclosure, IFA is used in murine studies,whereas alum or squalene is used in human studies. In instances where itis desirable to deliver hybrid WHcAg particles in a single dose insaline, a molecular adjuvant is employed. A number of molecularadjuvants are employed to bridge the gap between innate and adaptiveimmunity by providing a co-stimulus to target B cells or other APCs.

B. Other Molecular Adjuvants

Genes encoding the murine CD40L (both 655 and 470 nucleic acid versions)have been used successfully to express these ligands at the C-terminusof WHcAg (See, e.g., WO 2005/011571). Moreover, immunization of micewith hybrid WHcAg-CD40L particles results in the production of higheranti-core antibody titers than does the immunization of mice with WHcAgparticles. However, lower than desirable yields of purified particleshave been obtained. Therefore, mosaic particles containing less than100% CD40L-fused polypeptides are produced to overcome this problem. Theother molecular adjuvants inserted within the WHcAg, including the C3dfragment, BAFF and LAG-3, have a tendency to become internalized wheninserted at the C-terminus. Therefore tandem repeats of molecularadjuvants are used to resist internalization. Alternatively, variousmutations within the so-called hinge region of WHcAg, between theassembly domain and the DNA/RNA-binding region of the core particle aremade to prevent internalization of C-terminal sequences. However,internalization represents a problem for those molecular adjuvants suchas CD40L, C3d, BAFF and LAG-3, which function at the APC/B cellmembrane. In contrast, internalization of molecular adjuvants such asCpG ODN is not an issue as these types of adjuvants function at thelevel of cytosolic receptors.

Another type of molecular adjuvant or immune enhancer is the inclusionwithin hybrid core particles of a CD4⁺ T cell epitope, preferably a“universal” CD4⁺ T cell epitope that is recognized by a large proportionof CD4⁺ T cells (such as by more than 50%, preferably more than 60%,more preferably more than 70%, most preferably greater than 80%), ofCD4⁺ T cells. In one embodiment, universal CD4⁺ T cell epitopes bind toa variety of human MHC class II molecules and are able to stimulate Thelper cells. In another embodiment, universal CD4⁺ T cell epitopes arepreferably derived from antigens to which the human population isfrequently exposed either by natural infection or vaccination [29]. Anumber of such universal CD4⁺ T cell epitopes have been describedincluding, but not limited to: Tetanus Toxin (TT) residues 632-651; TTresidues 950-969; TT residues 947-967, TT residues 830-843, TT residues1084-1099, TT residues 1174-1189 [30]; Diphtheria Toxin (DT) residues271-290; DT residues 321-340; DT residues 331-350; DT residues 411-430;DT residues 351-370; DT residues 431-450 [31]; Plasmodium falciparumcircumsporozoite (CSP) residues 321-345 and CSP residues 378-395 [32];Hepatitis B antigen (HBsAg) residues 19-33 [33]; Influenza hemagglutininresidues 307-319; Influenza matrix residues 17-31 [34]; and measlesvirus fusion protein (MVF) residues 288-302 [35].

Methods of Inducing an Immune Response

The invention includes methods for eliciting an immune response in asubject in need thereof, comprising administering to the subject aneffective amount of an antigenic composition comprising one or more ofthe peptides, proteins, or VLP described herein. Also provided aremethods for eliciting an immune response in a subject in need thereof,comprising administering to the subject an effective amount of anantigenic composition comprising a polynucleotide encoding a chimericpeptide described herein, wherein said chimeric polypeptide expressed invivo assembles as a hybrid VLP. Unless otherwise indicated, theantigenic composition is an immunogenic composition.

The immune response raised by the methods of the present disclosuregenerally includes an antibody response, preferably a neutralizingantibody response, antibody dependent cell-mediated cytotoxicity (ADCC),antibody cell-mediated phagocytosis (ADCP), complement dependentcytotoxicity (CDC), and T cell-mediated response such as CD4*, CD8*. Theimmune response generated by the chimeric peptides, proteins, or VLPs asdisclosed herein generates an immune response that recognizes, andpreferably ameliorates and/or neutralizes, Zika virus. Methods forassessing antibody responses after administration of an antigeniccomposition (immunization or vaccination) are known in the art and/ordescribed herein. In some embodiments, the immune response comprises a Tcell-mediated response (e.g., peptide-specific response such as aproliferative response or a cytokine response). In preferredembodiments, the immune response comprises both a B cell and a T cellresponse. Antigenic compositions can be administered in a number ofsuitable ways, such as intramuscular injection, subcutaneous injection,intradermal administration and mucosal administration such as oral orintranasal. Additional modes of administration include but are notlimited to intranasal administration, intra-vaginal, intra-rectal, andoral administration. A combination of different routes of administrationin the immunized subject, for example intramuscular and intranasaladministration at the same time, is also contemplated by the disclosure.

Antigenic compositions may be used to treat both children and adults,including pregnant women. Thus a subject may be less than 1 year old,1-5 years old, 5-15 years old, 15-55 years old, or at least 55 yearsold. Preferred subjects for receiving the vaccines are the elderly(e.g., >55 years old, >60 years old, preferably >65 years old), and theyoung (e.g., <6 years old, 1-5 years old, preferably less than 1 yearold). Additional subjects for receiving the vaccines or compositions ofthe disclosure include naïve (versus previously infected) subjects,currently infected subjects, or immunocompromised subjects.

Administration can involve a single dose or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, or a mucosal prime and parenteral boost.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naive subjects or subjects of ahyporesponsive population (e.g., diabetics, or subjects with chronickidney disease (e.g., dialysis patients)). Multiple doses will typicallybe administered at least 1 week apart (e.g., about 2 weeks, about 3weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks,about 12 weeks, or about 16 weeks). Preferably multiple doses areadministered from one, two, three, four or five months apart. Antigeniccompositions of the present disclosure may be administered to patientsat substantially the same time as (e.g., during the same medicalconsultation or visit to a healthcare professional) other vaccines.

In general, the amount of protein in each dose of the antigeniccomposition is selected as an amount effective to induce an immuneresponse in the subject, without causing significant, adverse sideeffects in the subject. Preferably the immune response elicitedincludes: neutralizing antibody response; antibody dependentcell-mediated cytotoxicity (ADCC); antibody cell-mediated phagocytosis(ADCP); complement dependent cytotoxicity (CDC); T cell-mediatedresponse such as CD4*, CD8*, or a protective antibody response.Protective in this context does not necessarily require that the subjectis completely protected against infection. A protective response isachieved when the subject is protected from developing symptoms ofdisease, especially severe disease associated with the pathogencorresponding to the heterologous antigen. As described above, theimmune response generated by the chimeric peptides or VLP as disclosedherein generates an immune response that recognizes, and preferablyameliorates and/or neutralizes, Zika virus.

The WHcAg-ZIKV chimera vaccine administration and formulation may beoptimized to induce mucosal immune protection for preventing sexualtransmission. The invention contemplates mucosal route administrationsuch as nasal, vaginal, rectal or oral. The vaccine formulation can beoptimized using adjuvant/s formulation for stimulation of mucosal immuneresponse such as IgA and induction of mucosa-associated lymphoid tissues(MALTs). Adjuvants for mucosal immunization considered for WHcAg-ZIKVchimera vaccine include but are not limited to polymeric particles(e.g., Chitosan), cholera toxin (CT), and imidazoquinoline (Imiquimodand Resiquimod).

The WHcAg-ZIKV chimera vaccine formulation and administration may bedesigned to achieve a broader immune response for protection againstmultiple transmission routes: mosquito transmission, blood transfusion,maternal transmission, sexual transmission, organ transplant and otherpossible routes.

The amount of antigen (e.g., VLP) can vary depending upon whichantigenic composition is employed. Generally, it is expected that eachhuman dose will comprise 0.1-2000 μg of protein (e.g., chimericpeptide), such as from about 1 μg to about 2000 μg, for example, fromabout 1 μg to about 1500 μg, or from about 1 μg to about 1000 μg, orfrom about 1 μg to about 500 μg, or from about 1 μg to about 100 μg. Insome embodiments, the amount of the protein is within any range having alower limit of 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250μg, and an independently selected upper limit of 2000, 1950, 1900, 1850,1800, 1750, 1700, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300 or1250, 1200, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650,600, 550, 500, 450, 400, 350, 300 or 250 μg, provided that the lowerlimit is less than the upper limit. Generally a human dose will be in avolume of from 0.1 ml to 1 ml, preferably from 0.25 ml to 0.5 ml. Theamount utilized in an antigenic composition is selected based on thesubject population. An optimal amount for a particular composition canbe ascertained by standard studies involving observation of antibodytiters and other responses (e.g., antigen-induced cytokine secretion) insubjects. Following an initial vaccination, subjects can receive a boostin about 4-12 weeks.

Articles of Manufacture and Kits

The invention additionally includes articles of manufacture and kitscomprising a peptide, a chimeric peptide or protein, a fusion protein,or VLP described herein (FIGS. 12-14. In some embodiments, the kitsfurther comprise a solid support (e.g., referring to FIG. 12, the solidsupport can be a plate 1, a test strip 3, or a microbead 4). Kits orarticles also comprise, in some variations, a capture antibody 5 and/ora detection antibody 6. In some embodiments, the kits further compriseinstructions for measuring peptide-specific antibodies. In someembodiments, the antibodies are present in serum from a blood sample ofa subject immunized with an antigenic composition comprising the VLP.

Chimeric WHcAg-ZIKV VLP are designed for capturing anti-ZIKV and includebut are not limited to specific and selected amino acids sequence(s)from ZIKV viral protein E, NS1, prM/M or C (see Tables 1, 3, 5, and 7).Such recombinant amino acid sequences are inserted at a location betweentwo amino acids in the region of amino acids 77 to 82 of the WHcAgprotein (GenBank accession number NP_671816). See Table 2, 4, 6, and 8.

As used herein, the term “instructions” refers to directions orprotocols for using reagents contained in the kit for measuring antibodytiter. In some embodiments, the instructions further comprise thestatement of intended use required by the U.S. Food and DrugAdministration (FDA) in labeling in vitro diagnostic products. The FDAclassifies in vitro diagnostics as medical devices and required thatthey be approved through the 510(k) procedure. Information required inan application under 510(k) includes: 1) The in vitro diagnostic productname, including the trade or proprietary name, the common or usual name,and the classification name of the device; 2) The intended use of theproduct; 3) The establishment registration number, if applicable, of theowner or operator submitting the 510(k) submission; the class in whichthe in vitro diagnostic product was placed under section 513 of the FD&CAct, if known, its appropriate panel, or, if the owner or operatordetermines that the device has not been classified under such section, astatement of that determination and the basis for the determination thatthe in vitro diagnostic product is not so classified; 4) Proposedlabels, labeling and advertisements sufficient to describe the in vitrodiagnostic product, its intended use, and directions for use, includingphotographs or engineering drawings, where applicable; 5) A statementindicating that the device is similar to and/or different from other invitro diagnostic products of comparable type in commercial distributionin the U.S., accompanied by data to support the statement; 6) A 510(k)summary of the safety and effectiveness data upon which the substantialequivalence determination is based; or a statement that the 510(k)safety and effectiveness information supporting the FDA finding ofsubstantial equivalence will be made available to any person within 30days of a written request; 7) A statement that the submitter believes,to the best of their knowledge, that all data and information submittedin the premarket notification are truthful and accurate and that nomaterial fact has been omitted; and 8) Any additional informationregarding the in vitro diagnostic product requested that is necessaryfor the FDA to make a substantial equivalency determination.

As described herein, the invention also includes methods for screeninganti-Zika virus antibodies comprising: a) measuring binding of anantibody or fragment thereof to a VLP as described herein; and b)measuring binding of the antibody or fragment thereof to a WoodchuckHepatitis core Antigen protein (WHcAg) VLP devoid of a peptide asdisclosed herein; and c) determining that the antibody or fragmentthereof is specific or selective for a peptide of the disclosure whenthe antibody or fragment thereof binds to the chimeric VLP but not theWHcAg VLP devoid of a peptide of the disclosure. In some embodiments,the VLP is attached to a solid support. In further embodiments, thesolid support is a microbead, an assay plate, a test strip, or a filteras depicted in FIGS. 12 and 13. Methods for (i) screening anti-Zikavirus antibodies; (ii) detecting or measuring antibodies to Zika virusin a biological sample; or (iii) detecting a Zika virus infection mayall be performed using a solid support as shown in FIGS. 12, 13, and 14.In various embodiments, antigen-antibody complex formation and detectionmay be performed by attaching a VLP as described herein directly to asolid support (such as, e.g., a plate 1, a test strip 3, or a microbead4) and then contacting the VLP 7 with a test sample putativelycontaining an anti-Zika virus antibody 6 (see FIG. 12). Alternatively,or in addition, a VLP of the disclosure may be indirectly attached to asolid support by first attaching an anti-VLP antibody 5 to the solidsupport and then contacting the VLP 7 with the anti-VLP antibody to forma complex (see FIG. 12). A test sample putatively containing ananti-Zika virus antibody 6 is then applied, creating a “sandwich”complex between the anti-VLP antibody, the VLP, and the antibody fromthe test sample having an affinity for the VLP. Regardless of the methodchosen, detection of binding of an antibody from the test sample to theVLP is indicative of a Zika virus antibody being present in the sample.

Sandwich ELISA is used for detection of Zika Virus antibody in patients.The sandwich ELISA test for human Immunoglobulin G (IgG) is useful forthe detection of circulating long-lived, neutralizing anti-Zika virusantibody. The Immunoglobulin M (IgM) sandwich ELISA is very effectivefor the early onset of the infection when IgM response peaks. Using theELISA format, the wells of microtitre plates are coated with either Goatanti-human IgG or IgM, followed by incubation with subject serumcontaining anti-ZIKV antibodies in case of viral infection. Afterincubation, VLPs carrying Zika Virus peptide sequence are added to thewell, unbound antigen is washed out, and Horseradish Peroxidase (HRP)conjugated anti-Zika Virus monoclonal antibody (revealing monoclonalantibody) is added. The bound conjugate is detected after addition ofsubstrate solution such as TMB or enhanced chemiluminescence (ECL)reagent. The TMB reaction is terminated using stop solution and thedegree of substrate hydrolysis is measured using spectrophotometry platereader. Alternatively, the ECL signal can be detected using a platereader with luminometer detector right after the ECL substrate addition.

Early and accurate diagnosis of Zika Virus is very important, especiallyin the field. The Lateral Flow Immuno Assay (LFIA) is able to detectanti Zika Virus antibodies in sera from clinically proven patients, aswell as in healthy control subjects. The LFIA is used to detect subjectserum antibody against Zika Virus antigen. Colloidal gold particlelabelled goat anti human IgG/IgM (e.g., 1.0 mg/L) is used as thedetector reagent. Recombinant VLP protein (e.g., 1.0 mg/L) is capturedin the strip by anti-WHcAg antibody or absorbed directly to the support.Rabbit anti-goat IgG (1.0 mg/L) are immobilized in test and controllines, respectively, on a nitrocellulose membrane, acting as the capturereagents (FIGS. 13 and 14).

EXAMPLES

As described herein, the present disclosure is related to chimeric VLPscontaining and displaying epitopes and antigen from ZIKV. The disclosurealso provides methods for creation and production of such chimeric VLPsto their applications, including but not limited to vaccines,diagnostics, clinical studies, assay development and antibody discovery.The recombinant and chimeric WHcAg VLP function as a carrier for highlyimmunogenic and optimized amino acids sequence(s) from the Domain III ofthe E protein (E DIII) or other immunogenic sequences from E protein ofZIKV. In addition, chimeric WHcAg VLP may include specific and selectedamino acids sequence(s) from ZIKV viral protein NS1, prM/M or C (seeTables 1, 3, 5, and 7). Such recombinant amino acid sequences areinserted at a location between amino acids 77 and 82 of the WHcAgprotein (GenBank accession number NP_671816). See Tables 2, 4, 6, and 8.

The disclosure also provides optimized production and purification ofrecombinant WHcAg chimeric VLPs in Yeast cellular system: Komagataellaphaffii Kurtzman (ATCC® 76273™). The WHcAg chimera constructs aresubcloned in pD912 vector from ATUM (formerly DNA2.0) (available on theworld wide web at atum.bio) with secretion alpha-factor signal(SS_Alphafactor) linked to the N terminus of the WHcAg chimera sequence(FIG. 2). Alternatively, the WHcAg chimeric construct is inserted inpD902 vector without a secretion signal for cytosolic protein expressionand accumulation. Vector is linearized and used for creating highexpressing yeast clones by transformation or electroporation in yeastcells. Yeast clones are selected using Zeocin resistance marker insemi-solid culture (YPD Agar). WHcAg chimera protein expressioninduction is obtained by optimized culture and using methanolsupplementation. The secreted VLPs are purified from the yeast culturemedia by biochemical methods such as precipitation, ultracentrifugation,ultrafiltration, chromatography, tangential flow filtration (TFF) or acombination of such methods. VLPs that are expressed and accumulated inthe yeast cytosol (not secreted) are purified by cell lysis methods(physical and chemical) followed by precipitation, ultracentrifugation,ultrafiltration, chromatography, tangential flow filtration (TFF) or acombination of such methods.

Cimica et al. [16] have developed a VLP vaccine for RespiratorySyncytial Virus (RSV) at TechnoVax Inc. (Tarrytown, N.Y.). RSV-likeparticle (RS-VLP) vaccine was assembled with human metapneumovirus(hMPV) matrix protein as the structural particle scaffold, and RSVfusion glycoprotein (F) as the main immunogen. Structural vaccinologywas applied for increasing and optimizing F protein immunogenicity;multiple F constructs were generated and tested in antigenicallydifferent conformations. The immunization with RS-VLP vaccine adjuvantedwith the squalene-based emulsion afforded full protection and was safein the mouse model of RSV disease [16]. The present disclosure utilizedan alternative approach for the creation and production of ZIK-VLP. VLPscan be produce in large scale fermentation of Pichia pastoris cultureform selected clones. VLP purification is performed using state of theart methods such as: precipitation, ultracentrifugation, Tangential FlowFiltration (TFF), ultrafiltration and chromatography. Purity and qualityof ZIK-VLPs chimera is tested by immunoassays and electron microscopy.

Example 1 Early Development of a ZIKV VLP Candidate

The ZIKV Envelope (E) protein is a primary target for vaccinedevelopment because it displays epitopes able to induce neutralizing andprotective antibody in the host [36]. The E protein comprises themajority of the flavivirus surface and plays multiple roles in viralinfection: host receptor recognition and binding, membrane fusion, viralrelease from endosomal compartment, virion assembly, and egress. TheZIKV shell is assembled with 180 copies of the E protein and comprisesthe majority of the virion surface [23, 37]. The E protein of anyflavivirus including ZIKV shows a highly conserved structure that isdivided into three domains: Domain I (DI) consisting of a centralbeta-barrel domain; Domain II (DII) important for dimerization andvirion assembly; and Domain III (DIII) characterized by animmunoglobulin-like segment. Noticeably, the distal part of the DIIcontains a Fusion Loop domain with very high amino acid sequenceidentity between flavivirus.

Several studies in flavivirus including ZIKV have demonstrated that theE protein DIII (EDIII) is a primary antigenic target of specificneutralizing antibodies [38, 39]. In particular, it was shown thatstructural domains inside DIII can induce highly neutralizing andprotective antibodies in a mouse model [38]. The ZIKV Fusion Loop domainin DII can induce highly neutralizing antibodies [18] that are able tocross react with other flavivirus. Cross-reacting antibodies, however,have been demonstrated to induce antibody-dependent enhancement (ADE) ofZIKV infection in patients with a history of DENV infection [40]. TheFusion Loop domain is implicated in ADE effects of ZIKV infection [41].For these reasons, the present disclosure describes the use of a ZIK-VLPvaccine using EDIII selected epitopes as immunogen targets forneutralizing ZIKV.

Structural vaccinology was utilized for selecting specific epitopes andantigens from ZIKV EDIII (FIG. 3 and Table 5). Antigenic sequences fromZika Virus Envelope (E) protein were identified using the Cn3D softwarefrom NIH (available on the world wide web atncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml) for structural analysis, andthe CLC Sequence Viewer Qiagen (available on the world wide web atqiagenbioinformatics.com/products/clc-sequence-viewer/) for analysis ofacid sequence conservation and specificity between flavivirus. Thesequencing and structural data was obtained from US National Library ofMedicine National Institutes of Health (available on the world wide webat ncbi.nlm.nih.gov/pubmed). Using recombinant DNA technology, such EDIII epitopes were included in the Woodchuck Hepatitis core Antigen(WHcAg) scaffolding system for delivery of the epitopes (FIG. 3).

TABLE 17 DNA Constructs for production of ZIKV-VLPs CONSTRUCTS GENERATEDTESTED 1 WHcAg (MOCK CONTROL FOR IMMUNIZATION) YES 2 WHcAg CHIMERA EPROTEIN DOMAIN III FULL LENGTH YES 3 WHcAg CHIMERA E PROTEIN DOMAIN III,A-B LOOP YES 4 WHcAg CHIMERA E PROTEIN DOMAIN III, CX-C-D-DX LOOP YES 5WHcAg CHIMERA E PROTEIN DOMAIN III, DX-E LOOP YES 6 WHcAg CHIMERA EPROTEIN DOMAIN III, F-G LOOP YES 7 E PROTEIN (POSITIVE CONTROL FORIMMUNIZATION) YES

Such a system has been used successfully for vaccine candidates: humanHBV surface protein (HBsAg) or HBV core antigen protein (HBcAg) arecurrently in clinical trials for influenza virus and the malariaparasite (Plasmodium falciparum) [42]. Although the HB-VLP system is avery efficient platform for antigen delivery to APCs and B cells [43],such technology has two limitations: i) HBV proteins may not assembleproperly because the steric hindrance of the carried antigen; ii)preexisting immunity against HBV may reduce greatly the immunizationefficiency. For these reasons, we will adopt the WHcAg scaffoldingsystem [21] that was successfully applied for developing VLP-basedvaccines for RSV [44], and malaria parasite [45]. The WHcAg has theability to function as a carrier for a selected epitope/antigen peptide(e.g., 5-100 amino acids) for inducing a very specific antibodyresponse. Applying structural vaccinology, we have designed ZIKVDill-optimized antigens comprising either full length DIII domain, orselected DIII structural domains comprising the A-B loop, C-D loop, D-Eloop and F-G loop (Table 1 and 2, FIG. 3). Using recombinant expressiontechnology, DNA constructs for ZIK-VLP expression in Pichia pastoriswere developed, and the potential vaccine candidates are tested forefficacy and safety in an A129, AG129 and C57BL/6 treated withanti-IFNAR1 antibodies mouse model for ZIKV infection.

Example 2 Production of ZIK-VLP Using the Pichia Expression System

Appropriate Pichia yeast strains for protein expression are availablefrom, e.g., ATCC (e.g., Komagataella phaffii Kurtzman ATCC 76274™ orKomagataella pastoris ATCC® 76274™). Using in silico analysis,codon-optimized DNA constructs expressing the ZIKA EDIII antigensconserved between different strains were designed (FIGS. 2, 3A, and 3B).Constructs using the promoter from the Pichia alcohol oxidase 1 (AOX1)gene were developed to drive production of the recombinant protein(ATUM.bio). Purification of VLPs by ultracentrifugation andultrafiltration methods and assays for quantification, purity andimmunogenicity of the VLPs has been established [16]. Importantly, VLPsmorphology and purity was assessed using Electron Microscopy analysis(FIG. 5). Antigenicity of VLPs was tested using Western blotting and dotblot methods using different commercially available and testedcommercially available monoclonal antibodies against EDIII domain suchas ZV-2 (ATCC BEI Resources NR-50414 Monoclonal Anti-Zika Virus Envelope(E) Protein) and ZV-54 (Millipore Sigma MABF2046, Anti-Zika VirusAntibody) (FIGS. 6 and 7).

Example 3 Immunizing Animals: Mouse Study

Safety is determined in the context of pregnant female BALB/c mice andin the context of 5 week old male and female mice. In both cases(n=10/concentration), three different concentrations (10 μg, 25 μg and50 μg) of WHcAg-ZIKV chimera VLP are injected intramuscularly. Asnegative controls, PBS and WHcAg VLPs without Zika virus antigen areinjected. To evaluate safety in the context of a prime-boost strategy,an independent set of animals (n=10/concentration) is injected at 3weeks post the initial vaccination event with the same concentration ofVLP as used in the prime vaccination. The animals are weighed daily andtheir morphological features and behavior (eating, drinking, mobility,social behavior) are recorded in comparison with the negative controlgroup. Terminally sacrificed animals are necropsied to assess grosstoxicity at the level of the internal organs including the spleen andthe liver. The spleen tissue is banked for B-cell assays. Inflammatoryload is evaluated in these animals at the end of the study. Following aterminal bleed, serum is obtained and utilized to quantify inflammatorymediators in circulation following the prime alone and the prime-booststrategy. The Aushon Multiplex Platform (Ciraplex, Aushon Biosystems) orLuminex system is used to simultaneously quantify the levels ofinflammatory mediators. Such assays allow an analysis of multiplecytokines and chemokines in serum and tissue in vaccinated animals.

Animal studies towards characterizing the WHcAg-ZIKV chimera VLP vaccineare performed using three lethal models for ZIKV infection: i) the A129mouse model [46]; ii) the AG129 mouse model [47]; and iii) the C57BL/6immunocompetent mouse model treated with Anti-IFNAR1 antibody [27] (FIG.15). The challenge experiments are carried out according to Rossi et alin AG126 mouse model: 3 week old mice are the most susceptible to ZIKVinfection while 5 week old mice showed signs of disease but recovered[46]. The 5 week old mice continued to maintain detectable viral load inthe serum that could be compared with the 3 week old mice. Typicalvaccination strategies require at least 2-3 weeks duration for the hostto mount an immune response. For the three week old mice, this requiresvaccination to be carried out immediately after birth. There areuncertainties regarding robustness of the immune system in a newbornanimal. To address these concerns, in the current study, the 5 week oldanimal are challenged with a prime immunization at week 1 after birthand a boost at week 4 after birth, followed by challenge in week 5. TheA129 is an immunocompromised animal model that could be unable torecapitulate the immunization response. For this reason, theimmunocompetent mouse model BALB/c treated with Anti-IFNAR1 antibody isincluded before ZIKV challenge. The comparison between the two models isrelevant to improve immunization strategies including vaccine dosage andformulation according to [27]. The challenge experiments are conductedusing Zika Virus FSS13025 Cambodia strain [46], the Puerto Rico strain(PRVABC59) and other strains available at ATCC BEI-Resources (availableon the world wide web at beiresources.org). Standardized assays for thequantification of this strain by plaque assay and quantitativereverse-transcriptase polymerase chain reaction (qRT-PCR) have beendeveloped. The prime-only, prime-boost vaccinated animals (at themaximum tolerated concentration of VLP with no apparent toxic outcomes)are challenged after vaccination by intra peritoneal challenge with aZika virus strain (e.g., PRVABC59) with 1×10⁴ plaque-forming units(PFU). The infected animals are monitored continuously for one week. Ifthere is no protection or suboptimal protection, the animals will showsymptoms of disease. The animals are monitored for signs of illnessincluding weight loss, hunched posture and ruffled fur and for signs ofsevere disease including tremors, lethargy and anorexia. The mortalityrate of vaccinated animals versus unvaccinated controls is quantified.At the end of the study period, survivors are sacrificed and samplescollected for follow up studies. All sacrificed animals are terminallybled and serum collected. The serum is subjected to analysis ofinflammatory mediators. In addition, the circulating viral load(infectious viral titers and genomic copy numbers) is quantified in allexperimental and control groups. The neutralization antibody titers aredetermined using the serum samples by plaque reduction neutralizationassay (PRNT assay). PRNT₅₀ and PRNT₈₀ titer values will be obtained bythe method described in the art [16, 48]. Necropsy is conducted on allanimals and spleen isolated for B cell activity studies (describedbelow). General gross morphological examination of other internal organsincluding the liver is conducted. As flaviviruses, in general,demonstrate a tropism to the liver, the viral load in the liver +/− VLPis quantified.

A group of 5 mice were immunized twice (prime and boost) with theplacebo control (WHcAg CTRL VLPs devoid Zika antigen) and the Zikavaccine candidate (WHcAg CD loop VLPs) by intramuscular injection. TheVLPs dosage was 10 μg adjuvanted with squalene-based oil-in-waternano-emulsion AddaVax (InvivoGen). Boost immunization was performed 14days after prime immunization. After 28 days the prime immunization,animals were conditioned for Zika Virus infection using the anti-IFNAR1antibody according to the literature protocol [27], see FIG. 16. Viralinfection was performed by intraperitoneal injection 1 day afteranti-IFNAR1 antibody treatment, using 10,000 plaque forming units (PFU)of Zika Virus Puerto Rico strain PRVABC59 (ATCC, BEI ResourcesNR-50240). Serum viremia was analyzed 3 days viral post-injection usingquantitative Real-Time PCR (qRT-PCR), with the instrument for Bio-RadCFX96 Touch™, and the kit Bio-Rad iTaq Universal SYBR Green kit (Catalog#172-5151), following the manufacturer's instructions. Specific Zika PCRprimers used were according to the protocol of Lanciotti, R. et al.[49]: Forward oligo 5′ CCGCTGCCCAACACAAG 3′; and Reverse oligo 5′CCACTAACGTTCTTTTGCAGACAT 3′. Quantification of viral copy number permicroliter (μl) was obtained by standard curve approach using the ZikaVirus (strain PRVABC59) genomic RNA standard (ATCC, BEI ResourcesNR-50244). FIG. 17 shows that ZIKV copy number was decreased in the micereceiving the Zika vaccine candidate (WHcAg CD loop VLPs).

Safety is determined in the context of pregnant female BALB/c mice andin the context of 5 week old male and female mice. In both cases(n=10/concentration), three different concentrations (10 μg, 25 μg and50 μg) of VLP are injected intramuscularly. As negative controls, PBSand WHcAg VLPs without Zika virus antigen are injected. To evaluatesafety in the context of a prime-boost strategy, an independent set ofanimals (n=10/concentration) is injected at 3 weeks post the initialvaccination event with the same concentration of VLP as used in theprime vaccination. The animals are weighed daily and their morphologicalfeatures and behavior (eating, drinking, mobility, social behavior) arerecorded in comparison with the negative control group. Terminallysacrificed animals are further necropsied to assess gross toxicity atthe level of the internal organs including the spleen and the liver. Thespleen tissue is banked for B-cell assays. Inflammatory load isevaluated in these animals at the end of the study. Following a terminalbleed, serum is obtained and utilized to quantify inflammatory mediatorsin circulation following the prime alone and the prime-boost strategy.The Aushon Multiplex Platform (Ciraplex, Aushon Biosystems) or Luminexsystem is used to simultaneously quantify the levels of inflammatorymediators. Such assays allow an analysis of multiple cytokines andchemokines in serum and tissue in vaccinated animals. Zika-VLP vaccinecandidates are tested in a murine model for protection against fetaltransmission, assessing fetal viability, morphology and viremia. Thewell-established model for trans-placental transmission using the A129mouse is employed. In this model, infecting dams at embryonic day six(E6) results in placental insufficiency and fetal demise, while damsinfected at midstage E9 show cranial dimension reduction. Importantly,infection at E6 results in 100% nonviable fetuses, while infection at E9results in 90% fetal viability, 5 days after infection in both groups(see FIG. 16).

Mouse models will be useful for identify specific neutralizing antibodyagainst Zika Virus according to the literature [38].

Example 4 Cross-Reactivity for Zika Virus Antibody and Antigen DependentEnhancement Test

In vitro Assays for testing Antigen Dependent Enhancement (ADE) inZIK-VLP chimera vaccinated mice are performed for testing vaccinespecificity. Mouse serum from immunized animals with ZIK-VLP chimeravaccine is tested in a standard in vitro assay using U937 (ATCC®CRL-1593.2™) and K562 (ATCC® CCL-243™) lymphocyte cell-lines from ATCCaccording to methods known in the art. Briefly, serial dilutions ofheat-inactivated sera from BALB/c mice is incubated with DENV strainsfor each serotypes 1 to 4, for 1 hour at 37° C. As a positive controlfor ADE the pan-Flavivirus antibody, clone D1-4G2-4-15 (ATCC BEIResources, NR-50327) is also included. Serum from animals immunized withWHcAg VLPs will be used as a negative control.

The cells are incubated with the serum-virus mixture for 2 hours at 37°C. with multiplicity of infection (MOI) 3, and are washed in order toremove free viral particles. Viral titer in the culture supernatant ismeasured according to the art [27, 50] with standard quantitativeReal-Time-PCR (qRT-PCR) after 4 days, to allow for viral replication.

Example 5 Zika Virus Diagnostic

Antibody-sandwich ELISA. Antibody-sandwich ELISA is perhaps the mostuseful of the immunosorbent assays for detecting antigen/antibodybecause it is between 2 and 5 times more sensitive than thedirect/indirect ELISA in which antigen is directly bound to the solidphase. Two sets of sandwich ELISAs will be developed to 1) detect thepresence of long-lasting, neutralizing anti-Zika virus antibodies (IgG),and 2) enable early detection of anti-Zika IgM in clinical samples.

To detect ZIKV antigens in sandwich ELISA format, the wells ofmicrotitre plates are coated with antibody against the scaffoldingsystem WHcAg in order to capture different types of WHcAg-ZIKV chimeraVLPs. The ELISA plates are incubated with subject serum (human or mouse)containing anti-ZIKV antibodies. The bound conjugate is detected afteraddition of specific secondary antibody against IgG or IgM labeled withHorseradish Peroxidase (HRP). The detection of antibody against ZikaVirus antigen is performed using HRP substrates such as TMB or ECL and amicroplate reader instrument. A positive control using antibodygenerated against Zika Virus is included in the test, while negativecontrols include: WHcAg VLPs without any Zika antigen or not immunizedserum against Zika. The sandwich ELISA test for human IgG is useful forthe detection of circulating long-lived, neutralizing anti-Zika virusIgG. The IgM sandwich ELISA will be very effective for the early onsetof the infection when IgM response peaks.

Rapid Diagnostic Detection using Lateral Flow Immunoassay (LFIA) system.

Early and accurate diagnosis of Zika Virus is very important, especiallyon the field. The LFIA (FIG. 13) will be used to detect anti Zika Virusantibodies in sera from clinically proven patients, as well as inhealthy control subjects (FIG. 14). The lateral flow immunoassay (LFIA)is developed to detect subject serum antibody against Zika VirusEnvelope and NS1 antigen. Colloidal gold particle labelled goat antihuman IgG/IgM (1.0 mg/L) is used as the detector reagent. RecombinantWHcAg-ZIKV chimera VLP protein (1.0 mg/L) and rabbit anti-goat IgG (1.0mg/L) were immobilized in test and control lines, respectively, on anitrocellulose membrane, acting as the capture reagents. Alternativelyrecombinant WHcAg-ZIKV chimera VLPs can be captured on the support byimmobilized antibody able to bind the WHcAg scaffolding protein.

Example 6 Developing a Formulation of VLPs

Zika VLP vaccine is manufactured according cGMP guidelines andformulated following standard FDA guidelines. The vaccine is free fromadventitious agents and toxic chemicals. Formulations will includediluents, stabilizers, adjuvants and preservatives [12, 51]. The studiesdisclosed herein include formulation optimization in order to increasevaccine efficacy and safety.

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What is claimed is:
 1. A chimeric peptide comprising a first peptideselected from SEQ ID NOS: 2-11, 22-33, 46, 47, 50 and 51 operably linkedto a second heterologous peptide having an amino acid sequence that isat least 80% identical to a Woodchuck Hepatitis core Antigen protein(WHcAg) comprising an amino acid sequence of SEQ ID NO: 1 or afunctional fragment thereof.
 2. The chimeric peptide of claim 1 whereinthe first peptide replaces amino acids from positions 77 to 82 of SEQ IDNO: 1 or functional fragments thereof.
 3. The chimeric peptide accordingto claim 1 further including at least one peptide linker of 1-10 aminoacids linking the first peptide to the sequence that is at least 90%identical to a WHcAg protein.
 4. A polynucleotide comprising anucleotide sequence encoding the chimeric peptide of claim
 1. 5. Anexpression vector comprising the polynucleotide of claim 4 operablylinked to an expression control sequence.
 6. A recombinant host cellcomprising the expression vector of claim
 5. 7. The recombinant hostcell of claim 6, wherein the host cell is: (i) a eukaryotic cellselected from the group consisting of mammalian, yeast, insect, plant,amphibian and avian cells; or (ii) a prokaryotic cell.
 8. A virus likeparticle (VLP) comprising the chimeric peptide of claim
 1. 9. The VLPaccording to claim 8, attached to a solid support microbead, an assayplate, a test strip, or a filter.
 10. An antigenic compositioncomprising the VLP of claim 8, wherein the VLP is present in thecomposition at a concentration of about 0.1-2000 μg/ml, in apharmaceutically acceptable carrier, diluent, stabilizer, preservative,or adjuvant, said composition inducing one or more of a protectiveimmune response, production of anti-Zika neutralizing antibody, andproduction of anti-Zika protective antibody.
 11. An antigeniccomposition comprising the VLP of claim 8 in a pharmaceuticallyacceptable carrier, diluent, stabilizer, preservative, or adjuvant, saidcomposition comprising SEQ ID NO:
 15. 12. The antigenic composition ofclaim 11, comprising one or more VLPs comprising different sequencesselected from the group consisting of amino acid sequences at least 80%identical to SEQ ID NOs: 2-4, 6-11, 22-33, 46-47, or 50-51.
 13. Acomposition comprising the vector of claim 5 in a pharmaceuticallyacceptable carrier, diluent, stabilizer, preservative, or adjuvant. 14.The composition of claim 13, comprising an adjuvant.
 15. A kitcomprising the VLP of claim 8 packaged with at least one reagentselected from an enzyme substrate, a detection antibody, and a blockingbuffer.
 16. A vaccine comprising the antigenic composition of claim 11,and an adjuvant.
 17. A method of producing an immune response to a Zikavirus in a subject, comprising administering to the subject an effectiveamount of the antigenic composition of claim 11, thereby producing animmune response to a Zika virus in the subject.
 18. A method ofinhibiting Zika virus infection in a subject comprising administering tothe subject an effective amount of the vaccine of claim 16, therebypreventing a disease or disorder caused by a Zika virus infection in thesubject.
 19. The method of claim 18, wherein the administering isvaginal or nasal mucosal administration.
 20. A method of detecting ormeasuring antibodies to Zika virus in a biological sample comprising: a)contacting the VLP of claim 8 with a biological sample under conditionssuitable for the formation of an antigen-antibody complex; and b)measuring or detecting antibodies to Zika virus by detecting ormeasuring an antigen-antibody complex formed between antibodies in thebiological sample and the VLP.